Mechanistic studies on the interaction of reduced cobalamin (vitamin B12r) with nitroprusside

Published on Web 01/08/2003

Mechanistic Studies on the Interaction of Reduced Cobalamin

(Vitamin B12r) with Nitroprusside

Maria Wolak,^†^,^‡Grazyna Stochel,*^,^†and Rudi van Eldik*^,^‡

Faculty of Chemistry, Jagiellonian UniVersity, 30060 Krakow, Poland, and

Institute for Inorganic Chemistry, UniVersity of Erlangen-N u¨ rnberg, Egerlandstr. 1,

91058 Erlangen, Germany

Received August 14, 2002 ; E-mail: vaneldik@chemie.uni-erlangen.de

Abstract: The electron-transfer reaction between reduced cobalamin (Cbl(II)) and sodium pentacyano-

nitrosylferrate(II) (sodium nitroprusside, NP), as well as the subsequent processes following the electron-

1

transfer step, were investigated by spectroscopic (UV-vis, H NMR, EPR), electrochemical (CV, DPV)

and kinetic (stopped-flow) techniques. In an effort to clarify the complex reaction pattern observed at

physiological pH, systematic spectroscopic and kinetic studies were undertaken as a function of pH (1.8-

9) and NP concentration (0.0001 - 0.09 M). The kinetics of the electron-transfer reaction was studied

under pseudo-first-order conditions with respect to NP. The reaction occurs in two parallel paths of different

order, viz. pseudo-first and pseudo-second order with respect to the NP concentration, respectively. The

contribution of each path depends on pH and the [NP]/[Cbl(II)] ratio. At low pH and total NP concentration

I

+ -

(

pH < 3, [NP]/[Cbl(II)] ≈ 1), the cyano-bridged successor complex [Cbl(III)-(µ-NC)-Fe (CN)

3 s

(NO )] (1 ) is

the final reaction product formed in an inner-sphere electron transfer reaction that is coupled to the release

of cyanide from coordinated nitroprusside. At higher pH, subsequent reactions were observed which involve

the attack of cyanide released in the electron transfer step on the initially formed cyano-bridged species,

I

+ 2-

4

and lead to the formation of Cbl(III)CN and [Fe (CN) (NO )] . The strong dependence of the rate and

mechanism of the subsequent reactions on pH is attributed to the large variation in the effective nucleophilicity

of the cyanide ligand in the studied pH range. An alternative electron-transfer pathway observed in the

II

+ 2-

presence of excess NP involves the reaction of the precursor complex [Cbl(II)-(µ-NC)-Fe (CN)

4

(NO )]

II

+ -

NO]³^-, as

(1

p

) with NP to give [Cbl(III)-(µ-NC)-Fe (CN) (NO )] (2) and reduced nitroprusside, [Fe(CN)

4 5

the initial reaction products. Analysis of the kinetic data allowed elucidation of the rate constants for the

inner- and outer-sphere electron-transfer pathways. The main factors which influence the kinetics and

thermodynamics of the observed electron-transfer steps are discussed on the basis of the spectroscopic,

kinetic and electrochemical results. A general picture of the reaction pathways that occur on a short (s)

and long (min to h) time scale as a function of pH and relative reactant concentrations is derived from the

experimental data. In addition, the release of NO resulting from the one-electron reduction of NP by Cbl(II)

was monitored with the use of a sensitive NO electrode. The results obtained in the present study are

discussed in reference to the possible influence of cobalamin on the pharmacological action of nitroprusside.

Introduction

We have developed an interest in the reactions of the oxidized

1

,2

and reduced forms of cobalamin with NO and selected NO-

donating compounds that are of physiological and phar-

macological importance. The purpose of these studies is to

investigate and understand in more detail the specific interactions

which could lead to the observed modification of the physi-

ological activity of NO and selected NO-donors. Although

several studies have indicated the possible biological significance

Cobalamin (vitamin B12, compare Figure 1) is a cobalt-

containing macrocyclic complex, which serves as a cofactor in

a number of cobalamin-dependent enzymatic processes. Equato-

rial positions in cobalamin are occupied by the corrin ring, which

is structurally related to the porphyrin ligand. The axial position

below the corrin ring (R site) is usually occupied by an

intramolecularly bound 5,6-dimethylbenzimidazole group. The

upper axial position (â site) in oxidized forms of cobalamin

3

-6

of such interactions,

their chemical nature has not been

(

containing a Co(III) center) can be occupied by different ligands

(1) Wolak, M.; Stochel, G.; Hamza, M.; van Eldik, R. Inorg. Chem. 2000, 39,

-

2018.

(typically H2O in aquacobalamin or CN in cyanocobalamin),

(

2) Wolak, M.; Zahl, A.; Schneppensieper, T.; Stochel, G.; van Eldik, R. J.

Am. Chem. Soc. 2001, 123, 9780.

whereas in the reduced form (containing a Co(II) center), the

complex is five-coordinate, with one of the axial positions

remaining vacant.

(

3) Kruszyna, H.; Magyar, J. S.; Rochelle, L. G.; Russell, M. A.; Smith, R. P.;

Wilcox, D. E.J. Pharmacol. Exp. Ther. 1998, 285, 665 and references

therein.

(4) Brouwer, M.; Chamulitrat, W.; Ferruzzi, G.; Sauls, D. L.; Weinberg, J. B.

Blood 1996, 88, 1857 and references therein.

†

Faculty of Chemistry, Jagiellonian University.

(5) Zheng, D.; Birke, R. J. Am. Chem. Soc. 2001, 123, 4637 and references

‡

Institute for Inorganic Chemistry, University of Erlangen-N u¨ rnberg.

therein.

1334

9

J. AM. CHEM. SOC. 2003, 125, 1334-1351

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

II

+ -

a stable binuclear complex [Cbl(III)-(µ-NC)-Fe (CN)4(NO )]

1

(2), ( eq 1)

K₁

+

II

+ 2-

[

Cbl(III)(H O)] + [Fe (CN) (NO )] {\ }

2

5

II

+ -

[

Cbl(III)-(µ-NC)-Fe (CN) (NO )] + H O (1)

4

2

(

2)

Consequently, the use of cobalamin as an antidote for potential

cyanide poisoning induced by nitroprusside has been questioned.

Although kinetic and spectroscopic investigations have already

provided a rather detailed description of the mechanism and

11,13

products of reaction 1,

an interesting aspect which concerns

the change in redox activity of nitroprusside due to the formation

of complex 2, has not been elucidated and will be addressed in

the present study. The main goal of the work presented here,

however, was to investigate the intrinsic reactivity of reduced

cobalamin, a cobalamin derivative occurring in vivo,¹⁴and

sodium nitroprusside. We report on the formation and subse-

quent thermal reactions of the cyano-bridged complex [Cbl(II)-

Figure 1. Molecular structure of reduced cobalamin, Cbl(II).

clarified. In our earlier studies, we reported on the kinetic and

mechanistic characterization of the reaction between reduced

cobalamin and NO. This reaction was shown to result in the

-

formation of a stable Cbl(III)(NO ) complex on a micro-

2

II

+ 2-

second time scale, and thus may contribute to the observed

(µ-NC)-Fe (CN)4(NO )] (1p) (a one-electron reduced form

of 2), and comment on its possible biological relevance in

inhibition of the physiological activity of NO in the presence

of cobalamin.

2

-

relation to release of NO from [Fe(CN)5NO] .

We now report spectroscopic and kinetic studies on the

reaction of reduced cobalamin with a well recognized hypo-

tensive agent sodium pentacyanonitrosylferrate(II), commonly

known as sodium nitroprusside (SNP). The therapeutic proper-

A purely chemical aspect of this work is related to the

chemistry of oligonuclear cyano-bridged M-CN-M′ com-

plexes. Structural and electronic features as well as redox

properties of such complexes have been extensively studied in

recent years with respect to intermetallic electronic coupling,

thermal and photoinduced electron transfer, and energy transfer

processes occurring in such systems. Several examples of Co-

(µ-NC)-Fe complexes have been prepared and examined in

2-

ties of the nitroprusside ion, [Fe(CN)5NO] (NP), depend on

the release of nitric oxide, which in this formally Fe(III) nitrosyl

+

is considered as a three electron donor (i.e., NO ) bound to the

low-spin Fe(II) center. Despite numerous studies on the chemical

reactivity and electronic properties of nitroprusside, the mech-

anisms that lead to the observed physiological activity of this

compound are not fully understood. Two possible pathways have

been suggested in this context, the first of which relates mainly

11,13,15-17

the context of electron-transfer processes.

These include

III

n

recent studies on the [(N)5Co -(µ-NC)-Fe (CN)5] and

II

III

n-1

[(N)5Co -(µ-NC)-Fe (CN)5]

complexes, where (N)5 de-

1

5,16

notes a pentadentate macrocyclic amine.

Spectroscopic and

II

+

2-

+

to the ability of [Fe (CN)5(NO )] to act as an NO donor in

reactions with intracellular nucleophiles (most importantly

kinetic studies presented here concern the redox and substitution

reactivity of the binuclear cyano-bridged complex formed

between two biologically relevant species, viz. Cbl(II) and

7

thiols). The other pathway of NO release is suggested to

2

-

proceed via the redox conversion of NP to its one- or two-

electron reduced forms by reductants present in mammalian cells

[Fe(CN)5NO] .

8,9

Experimental Section

(such as ascorbic acid or glutathione).

An important limitation of the practical pharmacological

Materials. Hydroxocobalamin hydrochloride (> 98%) and sodium

nitroprusside (∼99.0%) were obtained from Sigma. Other chemicals

used throughout this study were of analytical reagent grade. Solutions

were prepared from deionized (Millipore) water. Due to the marked

oxygen sensitivity of Cbl(II) and reduced NP, all solutions were

application of NP results from the fact that its metabolic

-

decomposition is accompanied by release of CN . In this

respect, cobalamin which strongly binds cyanide to form a

1

4

-1 10

nontoxic Cbl(III)CN complex (KCbl(III)CN ) 1.2 × 10 M ),

was suggested as a scavenger of cyanide released from

metabolized NP. Subsequent studies provided evidence that the

physiological action of NP is altered in the presence of

2

prepared and handled in the absence of O with the use of Schlenk

techniques. Oxygen-free nitrogen was used to deoxygenate the solutions.

Kinetic and Spectroscopic Measurements. UV-vis spectral changes

and slow kinetic measurements were performed in gastight cuvettes in

the thermostated (( 0.1 °C) cell compartment of a Shimadzu UV-

2001 spectrophotometer. Faster spectral changes, as well as single-

wavelength kinetic measurements, were recorded on an Applied

1

1,12

cobalamin,

and this effect was ascribed to the formation of

(

6) Zheng, D.; Birke, R. Inorg. Chem. 2002, 41, 2548 and references therein.

7) Oszajca, J.; Stochel, G.; Wasielewska, E.; Stasicka, Z.; Gryglewski, R. J.;

Jakubowski, A.; Cieslik, K. J. Inorg. Biochem. 1988, 69, 121, and references

therein.

8) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.; Janczuk, A.

J. Chem. ReV. 2002, 102, 1091, and references therein.

9) (a) Smith, J. N.; Dasgupta, T. P. Inorg. React. Mech. 2002, 3, 181. (b)

Smith, J. N.; Dasgupta, T. P. J. Inorg. Biochem. 2001, 87, 165, and

references therein.

(

(13) Stochel, G.; van Eldik, R.; Kunkely, H.; Vogler, A. Inorg. Chem. 1989,

28, 4314.

(

(14) (a) Pezacka, E. H. Biochim. Biophys. Acta 1993, 1157, 167. (b) Watanabe,

F.; Saido, H.; Yamaji, R.; Miyatake, K.; Isegawa, Y.; Ito, A.; Yobisui, T.;

Rosenblatt, D. S.; Nakano, Y. J. Nutr. 1996, 126, 2947.

(15) (a) Bernhardt, P. V.; Martinez, M. Inorg. Chem. 1999, 38, 424. (b)

Bernhardt, P. V.; Macpherson, B. P.; Martinez, M. Inorg. Chem. 2000, 39,

5203.

(

10) Lexa, D.; Saveant, J. M.; Zickler, J. J. Am. Chem. Soc. 1980, 102, 2654.

11) Butler, A. R.; Glidewell, C.; McIntosh, A. S.; Reed, D.; Sadler, I. H. Inorg.

Chem. 1986, 25, 970 and references therein.

(

12) (a) Li, C. G.; Rand, M. J. Clin. Exp. Pharmacol. Physiol. 1993, 20, 633.

(16) Martinez, M.; Pitarque, M. A.; van Eldik, R. Inorg. Chim. Acta 1997, 256,

51.

(17) Rosenhein, L.; Speiser, D.; Haim, A. Inorg. Chem. 1974, 13, 1571.

(

b) Jenkinson, K. M.; Reid, J. J.; Rand, M. J. Eur. J. Pharmacol. 1995,

2

75, 145.

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1335

A R T I C L E S

Wolak et al.

Photophysics SX-17MV stopped-flow spectrophotometer equipped with

a monochromator/PMT single wavelength detection system and a rapid

scan J&M diode array detector. Kinetic and spectroscopic measurements

were performed at 25 °C. Solutions of Cbl(II) were prepared by

chemical reduction of aquacobalamin with sodium formate under inert

atmosphere and subsequent dilution of the stock Cbl(II) solution with

deoxygenated buffer to the appropriate concentration. Acetic acid/

sodium acetate buffer (0.1 M) and Tris buffer (0.1 M, Sigma Chemicals;

tively at pH 7.4), be expected that the outer-sphere electron

transfer between Cbl(II) and [Fe(CN)5NO] is thermodynami-

2-

cally unfavorable, and the observed redox processes involve

an inner-sphere electron-transfer mechanism. The primary

reactions which should be considered include formation of the

II

precursor cyano-bridged complex [Cbl(II)-(µ-NC)-Fe (CN)4-

+

2-

(

NO )] (1p), the electron-transfer step, and subsequent reac-

tions which lead to decomposition of the binuclear species.

Indeed, these steps have been observed, and their occurrence

and rate depended on the selected experimental conditions. The

complicated reactivity pattern observed results from rather

complex redox and substitution chemistry of both cobalamin

and NP. This mainly includes the influence of the ligand

environment on the redox properties of the Co and Fe centers

pH adjusted with HClO

and 7 to 9, respectively. Careful adjustment of the pH of Cbl(II) and

NP solutions with deoxygenated HClO or NaOH was done under other

pH conditions. UV-vis/NIR spectra (200-3200 nm) of 1:1 Cbl(II)/

4

) were used to control pH in the range 4 to 5.5

4

-

4

NP mixtures (2 × 10 M) at pH 1.8 were recorded in acidified D

2

O

on a Cary 5000 spectrophotometer.

EPR measurements were performed at 120 K on a Bruker ESR 300

E spectrometer. Samples were prepared under inert atmosphere by

18

present in cobalamin and NP, respectively, as well as the

-

3

addition of a molar equivalent of NP to the Cbl(II) solution (1 × 10

M) obtained by reduction of Cbl(II) with HCOONa in a 90% MeOH/

0% H O mixture (pH of the Cbl(II) solution was carefully adjusted

with small amounts of concentrated HClO or NaOH prior to addition

of NP). Aliquots of the samples were transferred in a gastight syringe

observed instability of the initially formed reaction products

toward substitution by cyanide present in the reaction medium.

1

2

Due to the biological relevance of the studied reaction, our

first attempt was to perform spectroscopic and kinetic investiga-

tions of the observed redox process at physiological pH. These

initial studies were frustrated by a complicated pattern of UV-

vis spectral changes observed at this pH, from which the

occurrence of reaction steps involving consecutive equilibration

processes could be inferred. The absorbance changes resulting

from these processes overlapped to a great extent, such that the

identity and kinetic behavior of the individual species was

difficult to resolve. These issues, however, could be addressed

by a systematic analysis of reactivity patterns as a function of

pH (1.8-9) and nitroprusside concentration (0.0001-0.09M).

The UV-vis spectral changes recorded for equimolar mix-

tures of the mononuclear complexes in the pH range 1.8 to 9,

as well as additional spectroscopic and electrochemical meas-

urements in aqueous and methanolic solutions, greatly assisted

the resolution of the primary and secondary reaction steps which

occur at equimolar concentrations of the reactants. With this

information, it was then possible to interpret more complex

spectral data and analyze kinetic results obtained in the presence

of an excess of sodium nitroprusside, as described below.

4

to the evacuated quartz EPR sample tubes, rapidly frozen in liquid N

and sealed to prevent entry of air.

2

NMR spectra were recorded on a Bruker Avance DPX300 NB

spectrometer operating at 300.13 MHz. Samples were prepared by

-

3

chemical reduction of aquacobalamin (2 × 10 M) in deoxygenated

D

2

O followed by addition of 1 equiv of NP. The apparent pH (pHapp

)

pH meter reading ) pD - 0.4) was adjusted by addition of DCl or

NaOD prior to addition of NP, and carefully remeasured before

transferring the solutions to gastight NMR tubes. Chemical shifts were

referenced internally to trimethylsilyl propionate (TSP).

Electrochemical Measurements. Cyclic and differential pulse

voltammograms were recorded using an EG&G Princeton Applied

Research Model 263 potentiostat. All measurements were performed

at room temperature under nitrogen atmosphere in a VC-2 voltammetry

cell (Bioanalytical Systems) with a standard three-electrode configu-

ration. Pt wire and a glassy carbon disk electrode (3.0 mm diameter)

were used as counter and working electrodes, respectively. Due to the

marked adsorption of the products of the electrochemical processes,

the surface of the working electrode was polished with 0.05 µm alumina

suspension prior to every scan. All potentials are referenced to an

4

aqueous Ag/AgCl electrode (E° ) 0.222 V). NaClO solution (0.1 M)

adjusted with NaOH or HClO to the appropriate pH, or 0.1 M

phosphate buffer was used as a supporting electrolyte.

NO Electrode Measurements. The release of NO resulting from

the addition of Cbl(II) to NP was monitored with the Isolated Nitric

Oxide Meter (WPI Instruments, model ISO-NO) calibrated by the

standard chemical method described in the suppliers manual (generation

of NO from NaNO in the presence of excess KI at acidic pH).

2

Measurements were performed in the dark under oxygen-free condi-

tions.

4

Equimolar Concentrations of the Reactants. Spectroscopic

Observations at Low pH. Successive additions of nitroprusside

to a Cbl(II) solution at pH e 2.5 under nitrogen atmosphere

resulted in definite changes in the UV-vis spectrum of

19

cobalamin, with isosbestic points observed at 342, 385, and

4

95 nm, as presented in Figure 2. The plot of absorbance at

70 nm versus total NP concentration (inset in Figure 2)

indicates a stoichiometry of close to 1:1 at equimolar concentra-

tions of the reactants, and a high equilibrium constant (K .

3

-1

1

0 M ) for the observed reaction. The spectral changes

Results and Discussion

recorded with a diode-array detector on a stopped-flow instru-

ment indicated that formation of the product is relatively rapid

under the employed experimental conditions (t1/2 < 1 s). This

product is stable under inert atmosphere, but in the presence of

air is oxidized, giving a spectrum identical to that of [Cbl(III)-

Preliminary Observations. Preliminary UV-vis spectro-

scopic and kinetic observations showed that the reaction of

_c_o_b₍_I_I₎_a_l_a_m_i_n_w_i_t_h_[_F_e₍_C_N₎₅_N_O_]2- is complex. The nature and

rate of the overall process, as well as the observed intermediates

and ultimate reaction products, strongly depended on pH, the

relative concentrations of the reactants, and the nature of the

solvent used as reaction medium. It was also evident from these

observations that electron-transfer processes are operative in the

studied system. It can on the basis of the redox potentials of

the reacting species (-0.04 and -0.36 V vs Ag/AgCl for

(

18) Lexa, D.; Saveant, J. M. Acc. Chem. Res. 1983, 16, 235 and references

therein.

(19) Due to the strong absorption bands of the corrin ring in the UV-vis region,

cobalamin is a much more distinctive chromophore compared to the

nitroprusside ion. The intraligand electronic transitions of cobalamin are

very sensitive to the oxidation state of the cobalt centre and nature of the

ligands present in the axial positions. Consequently, UV-vis spectroscopy

provides a good way to characterize the cobalamin reaction products, but

not those of the cyanonitrosylferrate species.

2-

3-

Cbl(III)/Cbl(II) and [Fe(CN)5NO] /[Fe(CN)5NO] , respec-

1336 J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

Figure 2. Spectral changes observed for the reaction of Cbl(II) (8 × 10^-⁵M) with NP at pH 2.5. Inset: plot of the absorbance at 470 nm versus total NP

concentration.

Scheme 1

II

+

-

ascribed to the interaction of an unpaired electron with ⁵⁷Co.

The hyperfine coupling constant of the low-field signal is

significantly smaller than that observed for reduced cobalamin

(

µ-NC)-Fe (CN)4(NO )] (the spectral changes for this reac-

tion exhibit well-defined isosbestic points at 290, 340, 370, and

4

98 nm).

2

0

A decrease in absorbance at 313 and 470 nm, accompanied

at pH 2 (ca. 150 G ). It is, however, similar to that observed

by a significant absorbance increase at 352 and ca. 545 nm

resulting from the addition of NP to Cbl(II), suggests a

considerable shift of electron density away from the Co(II)

center in the cobalamin product. However, the significant

remaining absorbance at 313 and 480 nm, as well as the absence

in the g|| component of Cbl(II) in the presence of cyanide (80

1

0,20

G

). Although detailed analysis of the spectrum was not

performed, we conclude that the observed low-field component

may reflect the residual unpaired electron density remaining on

the cobalt center, as is also suggested by other spectroscopic

data.

1

of the H NMR spectrum (see NMR data presented below) and

oxygen sensitivity of the product, indicate that the cobalt center

partially retains its Co(II) character. Further evidence in support

of this statement comes from EPR measurements. The EPR

spectrum obtained after addition of NP to reduced cobalamin

at pH 2 (Figure S1, Supporting Information) differs entirely from

that of reduced cobalamin observed under similar experimental

On the basis of the spectroscopic observations we ascribe

the absorbance changes observed at low pH to the acid-catalyzed

formation of the cyano-bridged binuclear species, [Cbl(III)-

I

+ -

(µ-NC)-Fe (CN) (NO )] , (1 ). According to the reaction

3

s

sequence presented in Scheme 1, the first step of the reaction

involves coordination of NP to Cbl(II) which exists in this pH

2

0

18,21,22

conditions. A key feature of the spectrum is a strong EPR

signal in the high field region, which is nearly identical with

range in the base-off form.

Due to the high lability of the

Co(II) center in reduced cobalamin, this step is expected to be

very rapid. It was difficult to separate this step from the

subsequent reactions under the employed experimental condi-

tions. This could, however, be done for a model system

I

+ 2-

that observed for [Fe (CN)4(NO )] formed after an one-

electron reduction of NP in acidic medium. This signal is

accompanied by a much weaker but clearly observable com-

ponent that appears in the low-field range of the spectrum. The

spectral features of this component resemble to some extent

that exhibited by reduced cobalamin. In particular, a multi-line

hyperfine splitting pattern was observed, which could be

4

-

involving the reaction of reduced cobalamin with [Fe(CN) ].

6

4

-

Addition of [Fe(CN)₆] to Cbl(II) solution results in a small

but clearly observable change in the UV-vis spectrum of

reduced cobalamin (Figure S3, Supporting Information). In this

case, however, no subsequent electron transfer reactions occur,

(

20) (a) Bayston, J. H.; Looney, F. D.; Pilbrow, J. R.; Winfield, M. E.

Biochemistry 1970, 9, 2164. (b) Schrauzer, G. N.; Lee, L. P. J. Am. Chem.

Soc. 1968, 90, 6541.

(21) Lexa, D.; Saveant, J. M.; Zickler, J. J. Am. Chem. Soc. 1977, 99, 2786.

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1337

A R T I C L E S

Wolak et al.

such that the observed spectral changes can be unambiguously

ascribed to formation of [Cbl(II)-(µ-NC)-Fe (CN)5], accord-

ing to reaction 2

statistics of the substitution process that the cis isomer is the

main product of the substitution reaction outlined in Scheme

1. Accordingly, 1s is the main reaction product observed in

the studied system at low pH.

II

4-

2

8

K₃

II

4-

The postulated complex belongs to the class of oligonuclear

M-NC-M′ species. A thoroughly investigated property of this

class of compounds is the optically induced metal-to-metal

charge-transfer giving rise to the intervalency band in their UV-

vis-NIR spectra. Analysis of this optical transition according

to the Hush theory often provides useful information on the

thermodynamic free energy of the electron-transfer process, the

center to center electronic coupling (Hab), and the reorganiza-

tional energy (λ), which are related to the energy, intensity and

halfwidth of the MMCT band, respectively. In the present case,

no apparent peak which could be ascribed to a MMCT transi-

tion could be observed in the range 700-3200 nm. It is,

however, possible, that this band is covered by the intense

intraligand transitions of the equatorial cobalamin ligand, as

Cbl(II) + [Fe (CN) ]

{\ } [Cbl(II)-(µ-NC)-Fe(CN)₅]

6

(2)

Kinetic measurements showed that the rate of this process is

too fast to be measured by stopped-flow technique. Detailed

spectroscopic and kinetic measurements performed on the

Cbl(II)/NP system (described in more detail below) indicate that

formation of 1p results in a similar small spectral change and

occurs on a time scale comparable to that observed for reaction

2.

Coordination of nitroprusside to Cbl(II) results in a very large

negative shift in the redox potential of the cobalt center, such

that it becomes significantly more negative than that character-

izing the one-electron reduction of NP (electrochemical data

supporting this statement are reported below). This change in

potential provides the driving force for inner-sphere electron

transfer within the binuclear complex. Oxidation of the cobalt

center resulting from this process accounts for the substantial

change in the UV-vis spectrum of reduced cobalamin. EPR

spectra as well as other spectroscopic observations (see text

below) indicated that the simultaneous one-electron reduction

of the bound nitroprusside is accompanied by the release of

HCN to the solution. The latter process can be ascribed to the

release of cyanide from the trans position to the nitrosyl ligand

in reduced nitroprusside (compare Scheme 1), in analogy to the

III

was also suggested for the structurally related [(N5)Co -(µ-

II

-

NC)-Fe (CN)5] dimers with pentadentate macrocyclic amine

ligands.

15

Spectroscopic Observations in the pH Range 3-9. The

Cbl(III)-(µ-NC)-Fe (CN)3(NO )] complex is the long-lived

I

+ -

[

species only in the lower pH range studied (pH < 3). On

increasing pH, subsequent reactions leading to decomposition

of the initially formed oligonuclear species were observed. In

addition, significant changes occurred in the kinetics and

thermodynamics of the inner-sphere electron-transfer step. The

successive UV-vis spectral changes recorded for equimolar

mixtures of the reactants in the pH range 2.4-9.0 indicated that

the overall reaction proceeds via different pathways, the relative

importance of which strongly depends on pH. This can be seen

from a comparison of the time dependent spectral changes

presented in Figure 3. The respective absorbance/time plots

showing evolution of the absorbance at 540 nm reported in

Figure 4 provide a semiquantitative kinetic characterization of

primary and secondary reaction steps that occur at different pH.

The following noteworthy features of the studied reaction can

be concluded from these data.

3-

well-known reaction observed for [Fe(CN)5NO] at pH <

1

1,23-27

5

II

•

3-

+

[

Fe (CN ) (CN )(NO )] + H a

eq 4

ax

I

+ 2-

[Fe (CN ) (NO )] + HCN (3)

eq 4

It should be noted that formation of 1p may involve coordination

of nitroprusside through the cyanide located in the cis and trans

position to the nitrosyl ligand, as was also observed for the

+

11

reaction of [Cbl(III)H2O] with NP. The release of cyanide

from the cis isomer is expected to result in a rapid formation of

1. The absorbance/time plots recorded within 50 s after

mixing of the reactants at different pH (Figure 4a) indicate that

the magnitude of the spectral change observed in this time

domain (which directly reflects the inner-sphere electron transfer

1

s, whereas in the case of the trans isomer, it would lead to

I

decomposition of the precursor to Cbl(III)CN and [Fe (CNeq)4-

+

2-

(NO )] . We conclude from the product distribution observed

at low pH (see NMR data presented below) and from the

in 1 ) decreases on increasing pH. Consequently, the significant

p

spectral change observed upon mixing of the reactants at pH 2

(

22) Formation of the binuclear complex from base-off Cbl(II) may involve

2

gradually decreases to a much less distinctive spectral change

at higher pH (Figure 3c). The initial spectral change observed

under these pH conditions (pH 5-8) resembles that resulting

substitution of a very labile solvent molecule by nitroprusside or its

coordination at the vacant R position, thus leading to the formation of â-

and R-substituted products, respectively. Indeed, the time-resolved UV-

vis spectral changes and the respective absorbance/time plots obtained at

pH e 3 indicated the occurrence of two processes which exhibited a similar

pattern of spectral changes (as shown in Figure S2, Supporting Information),

but occurred at noticeably different rates. The contribution of the slower

process to the overall reaction could be clearly observed at pH < 2, but

became negligible at pH > 3, i.e., when the dimethylbenzimidazole ligand

occupies the R position in Cbl(II). It is, therefore, reasonable to assume

that the slower process reflects the formation and subsequent electronic

stabilisation of the R-substituted cyano-bridged species. However, due to

the fact that this reaction path occurs only at low pH and is of little relevance

to the present study, its nature was not studied in more detail.

23) (a) van Voorst, J. D. W.; Hemmerich, P. J. Phys. Chem. 1966, 45, 3914

and references therein. (b) Rao, D. N.; Cederbaum, A. I. Biochim. Biophys.

Acta 1996, 1289, 195 and references therein.

II

4-

from the reaction of reduced cobalamin with [Fe (CN)6] (i.e.,

II

formation of a cyano-bridged complex with a Co -(µ-NC)-

II

Fe electronic configuration).

(28) (a) NMR data reported in the literature suggest the formation of the trans

isomer as the main product of the reaction between aquacobalamin and

1

NP. However, theoretical analyses of the charge distribution in

II

+

2-

5

[Fe (CN) (NO )] indicate similar net charges on the cyanide ligands

27, 28b

(

located in the cis and trans position to NO,

with a slightly more

negative charge for the cis cyanide (values such as -0.844 and -0.822

(au) were recently reported for the terminal nitrogen atoms in cis and trans

2

7

24) Cheney, R. P.; Simic, M. G.; Hoffman, M. Z. Taub, I. A. Asmus, K. D.

Inorg. Chem. 1977, 16, 2187.

25) Glidewell, C.; Johnson, I. L. Inorg. Chim. Acta 1987, 132, 145.

26) Schwane, J. D.; Ashby, M. T. J. Am. Chem. Soc. 2002, 124, 6822.

27) G o´ mez, J. A.; Guenzburger, D. Chem. Phys. 2000, 253, 73, and references

therein.

cyanide, respectively ). This suggests similar nucleophilic reactivity of

axial and equatorial cyanides toward coordination to the cobalt center, such

that the product distribution will mainly be determined by the statistics of

the substitution process. (b) Estrin, D. A.; Baraldo, L. M.; Slep, L. D.;

Barja, B. C.; Olabe, J. A.; Paglieri, L.; Corongiu, G. Inorg. Chem. 1996,

35, 3897.

(

1338 J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

Figure 3. Time evolution of the UV-vis spectra for the reaction of Cbl(II) with NP at pH 3.3 (a), 5.5 (b), 7.4 (c) and 9.0 (d). Spectra recorded at time

-

4

intervals of 1 min. [Cbl(II)] ) [NP] ) 2.5 × 10 M.

Figure 4. Absorbance/time plots showing the evolution of the absorbance at 540 nm over short (a) and long (b) time scale recorded for equimolar concentrations

-

4

of Cbl(II) and NP (2.3 × 10 M) at different pH (indicated in the figure).

2

. Data presented in Figures 3 and 4b show that at pH g 3

two bands centered at 515 and 547 nm observed during the

slow reaction step, indicate conversion of the initially formed

cyano-bridged species to a fully oxidized cobalamin product

which could be identified as cyanocobalamin (Cbl(III)CN) on

the fast reaction steps are followed by subsequent reactions

which occur on a longer time scale (min to h). The magnitude

of the absorbance change resulting from these processes

increases on increasing pH. Significant changes in absorbance

at 313, 470, and 360 nm accompanied by the appearance of

1

the basis of its UV-vis and H NMR spectra. Figure 5 presents

1

the aromatic region of the H NMR spectra recorded as a

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1339

A R T I C L E S

Wolak et al.

I

+ 2-

formation of a new signal characteristic for the [Fe (CN)4(NO )]

23

ion during the induction period (compare Figure S4, Support-

ing Information). It follows that the induction periods directly

reflect electron transfer between the cobalt and iron center at

pH > 4. This process is apparently accompanied by the release

of cyanide, as clearly evidenced by subsequent reactions

involving the attack of cyanide on the initially formed cyano-

bridged species.

4

. The time evolution of the UV-vis spectra recorded at pH

>

6 indicate an increasing contribution of a new reaction path

I

which leads to the formation of Cbl(III)CN and [Fe (CN)4-

+

2-

1

(NO )] (as evidenced by UV-vis, H NMR, and ERP

spectroscopy) through intermediates different from that observed

at lower pH. This can be seen on comparing the UV-vis spectra

shown in Figure 3c and 3d, and the respective kinetic traces

obtained at pH > 6 presented in Figure 4b. The characteristic

pattern of UV-vis spectral changes featuring the additional

reaction path, consisted of the appearance and subsequent slow

decay of a shoulder in the range 560-600 nm. Occurrence of

such a band in the low energy region of the spectrum indicates

formation of cobalamin species in which the cyanide ion is

3

coordinated at the R site of the corrin ring. Formation of

R-cyano substituted species could be clearly observed at

physiological pH, and became the predominant reaction path

at pH g 8. Rapid scan measurements performed with a diode-

array unit of a stopped-flow instrument allowed closer examina-

tion of this reaction path. The time evolution of the UV-vis

spectra at pH 8.0 shown in Figure 6 allows resolution of two

reaction steps within the time scale of the experiment (the final,

third step occurs on a significantly longer time scale). The first

step is evidenced by a characteristic initial spectral shift and

subsequent small spectral changes observed as an induction

period in the corresponding absorbance/time plot. We conclude

on the basis of the UV-vis and EPR spectroscopic observations

that this step involves formation of 1p, followed by electron

transfer and release of cyanide. These processes, however, occur

initially in a rather small fraction of the precursor complex (see

text below). The second reaction step that results in a large

spectral change indicates quantitative conversion of the initially

formed cyano-bridged species to the R-cyano substituted co-

balamin. This process is characterized by isosbestic points at

Figure 5. Aromatic region of the ¹H NMR spectra recorded after the

-

3

reaction of equimolar concentrations of cob(II)alamin and NP (2 × 10

M) at pHapp: 2.0 (a), 3.1 (b), 3.5 (c), 4.0 (d), 4.5 (e), 5.1 (f), 5.5 (g), 6.0

h), and 6.9 (i).

(

function of pHapp for equimolar mixtures of Cbl(II) and

-

[

Fe(CN)5NO]²ca. 3 h after mixing of the reactants. At pHapp

)

2, weak signals attributable to small amounts of Cbl(III)CN

II

+ -

and [Cbl(III)-(µ-NC)-Fe (CN)4(NO )] (2) were observed,

the main cobalamin product being NMR silent.²⁹On increasing

pH, the intensity of the signals at 6.06, 6.28, 6.58, 7.14, and

7

.26 ppm characteristic for Cbl(III)CN³⁰increased, indicating

a gradual increase in the conversion of the paramagnetic

cobalamin species to cyanocobalamin.³¹NMR and UV-vis data

indicated complete conversion of the cyano-bridged species to

cyanocobalamin at pH > 5, whereas at lower pH an equilibrium

mixture of 1s and Cbl(III)CN was observed.

3

. Formation of cyanocobalamin in the final reaction step

3

90, 492, and 610 nm. The R-cyano substituted intermediate

was preceded by characteristic induction periods which could

undergoes subsequent slow isomerization to cyanocobalamin,

with isosbestic points at 576, 502, 370, 502, 370, 342, and 287

nm (compare Figure 3d). The rate of this process is strongly

retarded on increasing pH, as evidenced by the final sections

of the kinetic traces recorded at pH > 6 which are presented in

Figure 4b.

be clearly distinguished from other reaction steps at pH > 4.

2

-

EPR studies performed for equimolar Cbl(II) and [Fe(CN)5NO]

3

2

concentrations in a 90% MeOH/10% H2O mixture at pH 7.4

indicated a decay of the Co(II) signal of cob(II)alamin and

(

29) At pH 2 very weak signals were also observed at ca. 9.2, 7.6, 7.5, and

6

.65 ppm. Although these signals clearly indicate formation of the base-

Scheme 2 summarizes the main reactions suggested to account

for the spectral evolution observed on a short (s) and long (min

to h) time scale in the pH range 3-9.0. Due to the fact that

off dimethylbenzimidazole-protonated cobalamin species, the low intensity

and broad features of the signals do not allow their unambiguous

assignment.

(

30) Calafat, A.; Marzilli, L. J. Am. Chem. Soc. 1993, 115, 9182.

I

+

2-

18,21

(

31) Formation of [Fe (CN)

4

(NO )] as the other reaction product was

reduced cobalamin exists in the base-on form at pH > 3,

confirmed by EPR spectroscopy.

formation of the precursor complex in this pH range involves

rapid coordination of NP to the vacant â site of reduced

cobalamin and is followed by inner-sphere electron transfer.

Experimental results indicate, however, that the latter process

is strongly influenced by the thermodynamics of cyanide release

from the reduced nitroprusside moiety. In the case of free

(

32) (a) The UV-vis spectral changes and the respective absorbance/time plots

recorded in this medium at different pH (adjusted with small amounts of

4

NaOH or HClO ), were in general similar to those observed for the reaction

in water. The overall reaction time, however, and in particular the induction

periods preceding the formation of cyanocobalamin, were significantly

longer, such that this reaction step could be conveniently studied by

conventional EPR spectroscopy. Significant deceleration of the reaction

observed in methanolic medium can at least in part be ascribed to a decrease

in driving force for the electron-transfer reaction due to a negative shift of

3

2b

the redox potential of the iron center on going from water to methanol.

b) Baraldo, L. M.; Forlano, P.; Parise, A. J.; Slep, L. D.; Olabe, J. A.

Coord. Chem. ReV. 2001, 219, 881 and references therein.

(

(33) Hamza, M. S.; Zou, X.; Brown, K. L.; van Eldik, R. Inorg. Chem. 2001,

40, 5440, and references therein.

1

340 J. AM. CHEM. SOC. VOL. 125, NO. 5, 2003

9

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

Figure 6. Time evolution of the UV-vis spectra recorded at time intervals of 5 s for an equimolar mixture of the reactants at pH 8.0. Experimental

-

4

conditions: [Cbl(II)] ) [NP] ) 1 × 10 M, pH adjusted with NaOH. Inset: absorbance/time plot showing the evolution of absorbance at 540 nm.

Scheme 2

Fe(CN)5NO]³ion, the following kinetic and thermodynamic

-

(pKa

HCN

) 9.3 ( 0.2 ). At pH > 6, however, the release of

24

[

-

parameters that characterize the release of cyanide (reaction 4)

have been reported²⁴

CN becomes thermodynamically unfavorable, such that

3-

[Fe(CN)5NO] becomes the dominant form of reduced nitro-

prusside at basic pH. The pK values for the overall protonation

equilibrium (5), determined by different experimental tech-

2

3,24,34

niques, fall in the range 4.7-6.5

II

•

3-

+

I

+ 2-

[

Fe (CN) (NO )] + H a [Fe (CN) (NO )] + HCN

5

4

(5)

k4 ) 2.8 × 10 s , k-4 ) 4 × 10 M s^-¹and K4 ) 6.8 ×

2

-1

6

-1

In the presently studied system, much slower reaction rates were

observed for the electron-transfer coupled release of cyanide

from 1p over the whole pH range studied, as compared to that

-

5

1

0

M at 25 °C. Despite the small value of K4, equilibrium

4) is shifted to the right at pH < 4 due to protonation of cyanide

(

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1341

A R T I C L E S

Wolak et al.

3-

reported for [Fe(CN)5NO] . This emphasizes the different

nature of the rate-determining step that leads to the loss of

cyanide from bound and free reduced NP, as discussed in more

detail below.

The reactivity of the cyano-bridged complex formed at pH

>

5 involves spontaneous rather than proton-driven release of

-

24

CN , a process which is expected to be thermodynamically

3

-

unfavorable (compare small value of K4 for [Fe(CN)5NO] ,

reaction 4). Experimental data indicate that the thermodynamics

of cyanide loss strongly affects inner-sphere electron transfer

in the [Cbl(II)-(µ-NC)-Fe (CN)4(NO )] complex. The latter

apparently becomes thermodynamically less favored at pH >

II

+ 2-

2

4,34

5

, in comparison to that accompanying the proton-driven

release of cyanide at lower pH. In this context, the decrease in

the initial absorbance change (Figure 4a) can be traced to the

pH dependent equilibrium (6) determining the ratio of the 1p

and 1s species, which gradually shifts to the right on decreasing

pH

Figure 7. Time evolution of the UV-vis spectra recorded at pH 7.4 for

-

4

an equimolar mixture of the reactants, [Cbl(II)] ) [NP] ) 1 × 10 M, in

+

-4

the presence of Ag (2 × 10 M). Spectra recorded at time intervals of

0.01 s, overall reaction time 0.2 s; pH adjusted with Tris-HClO4 buffer

solution.

II

+

2-

+

[

Cbl(II)-(µ-NC)-Fe (CN) (NO )] + H a

4

Scheme 3

I

+ -

[

Cbl(III)-(µ-NC)-Fe (CN) (NO )] + HCN (6)

3

We conclude that the small initial change in absorbance

observed after mixing of the reactants at pH > 7 reflects

formation of the precursor complex 1p as the main initial

reaction product, with little contribution from electron transfer

to the absorbance change. From the magnitude of the initial

spectral change observed at 540 nm, an apparent pK value of

orbital of Fe.²⁷Similarities in the electrochemical properties of

bound and free nitroprusside (as indicated by electrochemical

data reported later in the text) imply that cyanide loss from 1p

will result in analogous changes in the energy and symmetry

of the HOMO orbital at the iron center. Such changes, however,

may strongly modify the bridge-mediated metal-metal interac-

tions which govern the inner-sphere electron-transfer process.

Theoretical data on the electronic configuration of reduced

nitroprusside outlined above suggest that cyanide release

influences reducibility of the iron center by enabling the facile

iron-centered reduction within the binuclear complex (electron

5

(

.7 was estimated for the overall protonation equilibrium (6)

compare data presented in Figure S5, Supporting Information).

This value lies in the range of pK values reported for equilibrium

(

5), indicating that acid-base properties of nitroprusside in

II

+ 2-

[Cbl(II)-(µ-NC)-Fe (CN)4(NO )] are not significantly dif-

3-

ferent from that observed for [Fe(CN)5NO] .

The important influence of the release of cyanide on inner-

sphere electron transfer observed in the present system was

directly confirmed by an experiment in which equimolar

II

2-

-4

transfer in the Co -(µ-NC)-Fe complex which does not

concentrations of Cbl(II) and [Fe(CN)5NO] (1 × 10 M)

were reacted in the presence of Ag ions ([Ag ] ) 2 × 10

M) at pH 7.4. The resulting spectral changes presented in Figure

closely resemble those observed at pH 2, and indicate rapid

t < 0.2 s) occurrence of inner-sphere electron transfer. This

-

+

-4

release CN would be expected to result in a ligand (NO) rather

than a metal-centered reduction of nitroprusside).

7

(

The subsequent slow reactions observed at pH > 3 result

from the attack of cyanide liberated in the electron-transfer step,

on the initially formed cyano-bridged species. This may occur

observation can be accounted for by rapid formation of 1s as

outlined in Scheme 3.

-

along two reaction pathways involving the attack of CN on

Recent studies on the electron-transfer process involving

reduction of NP by selected biological reductants indicated a

similar strong influence of a reversible cyanide release on the

the â- or R-site of cobalamin, respectively. The relative

contribution of both these pathways was observed to depend

strongly on pH. Clean conversion of the cyano-bridged species

to cyanocobalamin observed as the final reaction step in the

pH range 3-6 can be accounted for by the reaction paths A

and A′ depicted in Scheme 2. Attack of cyanide on the upper

(â) position of cobalamin in the cyano-bridged complexes 1_p

(path A) and/or 1s (path A′) leads to formation of a very stable

Cbl(III)CN complex and thus, strong stabilization of the 3+

oxidation state of the cobalt center. The significant increase in

the rate and degree of conversion of the cyano-bridged species

to cyanocobalamin on increasing pH apparently reflects the

9

thermodynamics of the electron-transfer reaction.

The effect of the release of the peripheral cyanide ligand on

the reducibility of the iron center in 1p can be rationalized in

terms of large differences in the electronic structure of the

-

2-

27

[

Fe(CN)5NO]³and [Fe(CN)4NO] ions. It has been reported

3

-

that cyanide loss from [Fe(CN)5NO] results in a drastic change

in its electronic configuration. In particular, the nature of the

SOMO orbital changes from that consisting primarily of the

π* orbital of NO (and to a lesser extent of the 3dxz and 3dyz

orbitals of Fe) and exhibiting π symmetry, to one of σ symmetry

in which the unpaired electron is localized mainly on the 3dz2

-

increase in reactivity of the CN ions toward substitution. The

observed induction periods preceding formation of cyano-

cobalamin indicate that cyanide ions, once released from the

cyano-bridged species, accelerate the electron-transfer step.

(

34) Masek, J.; Maslova, E. Collect. Czech. Chem. Commun. 1974, 39, 2141

and references therein.

1342 J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

Formation of the R-cyano intermediate observed at pH > 6

can be accounted for by a contribution of reaction path B which

involves attack of cyanide at the R-position of cobalamin in

path C of Scheme 2. A similar reactivity pattern has indeed

been observed for several organocorrinoids which undergo

dealkylation by cyanide in a single kinetic step to give

II

+

2-

33

[Cbl(II)-(µ-NC)-Fe (CN)4(NO )] (1p). It is, in principle,

Cbl(CN)2. Irrespective of the exact nature of the R-cyano

possible that the R-cyano intermediate could be also formed

via attack of CN at the R-position in the successor [Cbl(III)-

substituted intermediate, its decomposition in a final, slow

reaction step observed at pH > 6 clearly involves the displace-

-

I

+

-

I

+ 2-

(

µ-NC)-Fe (CN)3(NO )] complex (1s). Very low concentra-

ment of [Fe (CN)4(NO )] coordinated to the cobalt center by

-

tions of CN present under the employed experimental condi-

tions imply, however, that the observed substitution of

R-dimethylbenzimidazole can only occur if the Co-N(dimethyl-

benzimidazole) bond is very labile. It can be expected that the

stabilization of the 3+ oxidation state of the cobalt center in 1s

CN released from the R-position of cobalamin, resulting in

I

+ 2-

formation of Cbl(III)CN and [Fe (CN)4(NO )] as the ultimate

reaction products. Two possible pathways (B-1 and C-1, Scheme

2) can be postulated for this process, depending on the actual

nature of the R-cyano intermediate.

35

would result in a stronger Co-N(dimethylbenzimidazole) bond

Literature data indicate that the cyanide ligand offers a greater

compared to that observed in 1p (containing the labile Co(II)

center). For this reason, the latter species is expected to be more

reactive toward substitution at the R-position.

stabilization of the 3+ oxidation state of the cobalt center in

10,18

cobalamin compared to dimethylbenzimidazole.

Thus, sub-

-

stitution of the DMBI ligand by CN upon formation of the

Attempts to characterize the R-cyano substituted intermediate

by recording H NMR spectra of the reaction mixture in basic

R-cyano intermediate is expected to promote electronic isomer-

1

ization of 1 , which in turn is coupled to the release of cyanide

p

D2O, were not successful due to a relatively rapid conversion

of the intermediates to cyanocobalamin under the experimental

conditions employed in the NMR measurements (pD ) 9.0,

(as shown in the reaction path B). The suggested reaction

sequence offers a further mechanistic interpretation of cyanide-

assisted inner-sphere electron transfer observed at high pH.

In the context of the reaction mechanism outlined in Scheme

2-

-3

[

Cbl(II)] ) [Fe(CN)5NO ] ) 2 × 10 M). It was, however,

possible to obtain such data when the reaction was carried out

in CD3OD at pD ) 9³⁶(adjusted with small amounts of NaOD).

The aromatic region of the H NMR spectrum recorded during

2

, the UV-vis spectral changes observed at physiological pH

indicate rapid formation of a mixture of [Cbl(III)-(µ-NC)-

1

I

+

-

II

+ 2-

Fe (CN)3(NO )] and [Cbl(II)-(µ-NC)-Fe (CN)4(NO )] (1p

and 1s, respectively) in which the latter is the main initial

product. The subsequent, slow reactions occurring at this pH

mainly involve reactions A and A′, with a minor contribution

of reaction sequence B to the overall process.

the entire course of the reaction (data not shown) exhibited five

intense peaks located at 7.25, 7.14, 6.58, 6.28(d), and 6.05 ppm,

which are attributed to cyanocobalamin formed as the final

cobalamin product (this was confirmed by recording the

spectrum of an authentic sample of cyanocobalamin in CD3OD

at pH 9). In addition, five peaks of lower intensity and

significantly broadened features were observed at 8.34, 7.41,

Excess of Nitroprusside. Spectroscopic Observations. To

investigate in more detail the kinetics and mechanism of the

observed electron-transfer process, kinetic studies under pseudo-

first-order conditions with respect to nitroprusside were under-

taken. The choice of the reactant used in excess was dictated

by the need to avoid experimental complications which arose

in the preliminary kinetic studies in the presence of excess

7

.38, 6.31, and 5.79 ppm, i.e., at the positions almost identical

with that observed for dicyanocobalamin, Cbl(CN)2, (peaks at

.36, 7.42, 7.38, 6.31, and 5.79 ppm, respectively) under the

8

same experimental conditions. This result confirms coordi-

nation of the cyanide ligand at the R-site of cyanocobalamin in

the observed intermediate. However, due to the fact that the

37

Cbl(II).

In analogy to the data obtained for equimolar concentrations

of the reactants, spectroscopic observations in an excess of NP

indicated the occurrence of fast and slow reaction steps, the

nature and rate of which strongly depended on pH. However,

the reactivity patterns were different from that observed under

1

H NMR spectrum of the R-cyano substituted complex

I

+ 2-

[(CN)Cbl(III)-(µ-NC)-Fe (CN)3(NO )] can be similar to that

of dicyanocobalamin (and in addition, is likely to exhibit

significantly broadened peaks, as was indeed observed in the

performed experiment), an unambiguous differentiation between

these two species could not be made. Because the concentrations

1:1 stoichiometric conditions. In particular, a significant increase

in the rate and in the degree of conversion of Cbl(II) to its

oxidized form in the initial, fast reaction steps was observed

on increasing the NP concentration. This is evidenced by the

initial sections (0-400 s) of the absorbance/time plots presented

in Figure 8. Spectroscopic data also indicated that [Cbl(III)-

-

of CN liberated in the electron-transfer step are very low,

formation of the mono-cyanide substituted [(CN)Cbl(III)-(µ-

I

+ 2-

NC)-Fe (CN)3(NO )] species rather than dicyanocobalamin

is a reasonable indirect conclusion. However, the trans-labilizing

effect of the cyanide ligand in the R-position may induce rapid

II

+ 2-

(

µ-NC)-Fe (CN)4(NO )] (2) rather than its reduced forms

I

+

2-

-

displacement of [Fe (CN)4(NO )] by a second CN , thus

1p and 1s, is the main cobalamin product formed in the electron-

leading to the formation of dicyanocobalamin, as suggested in

transfer step in the presence of excess NP. This observation

can be accounted for by the reactions outlined in Scheme 4.

The reaction route characterized by the rate constant k6

represents the contribution of outer-sphere electron transfer

between 1p and NP, to the overall process. Examination of the

products formed in the electron-transfer step as a function of

pH and NP concentration indicated that, in general, the

(

35) Significantly stronger Co-N(DMBI) bond in typical Cbl(III) derivatives

compared to Cbl(II) is evidenced by (i) shorter length of the Co-N(DMBI)

+

35b

35c

bond (1.925 Å in [Cbl(III)H

significantly lower pK

2 a

pK ) -2.1 in [Cbl(III)H O] compared to pK ) 2.9 in Cbl(II)). (b)

2

O]

compared to 2.13 Å in Cbl(II) ), (ii)

3

5d-e

a

values for deprotonation of the DMBI ligand

+

(

a

Kratky, C.; Farber, G.; Gruber, G.; Wilson, K.; Dauter, Z.; Nolting, H.-F.;

Konrad, R.; Krautler, B. J. Am. Chem. Soc. 1995, 117, 4654. (c) Krautler,

B.; Keller, W.; Kratky, C. J. Am. Chem. Soc. 1989, 111, 8936. (d) Brown,

K. L. J. Am. Chem. Soc. 1987, 26, 2034 and references therein. (e) Brown,

K. L.; Peck-Siler, S. Inorg. Chem. 1988, 27, 3548.

(

36) Despite a significantly slower rate of the reaction compared to that observed

in H

2

O, formation of the R-cyano substituted species in CD

3

OD was

(37) These mainly include the high extinction coefficients of Cbl(II), and

formation of cyano-bridged polynuclear species, such as Co-Fe-Co

trimers.

confirmed by a characteristic increase in absorbance in the range 580-

00 nm of the UV-vis spectrum.

6

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1343

A R T I C L E S

Wolak et al.

Scheme 4

pH conditions indicated that the cyanide-assisted electron-

transfer processes involving formation of the R-cyano substituted

intermediate (Scheme 2, reaction path B) are fast, and efficiently

compete with the outer-sphere electron-transfer reaction. The

final reaction step observed under neutral and basic pH

conditions (reflected by the increase in absorbance at 540 nm

in the final sections of the absorbance/time plots shown in Figure

8

) indicate slow conversion of the R-cyano intermediate to

cyanocobalamin. This process is expected to occur according

to the same reaction mechanism as that suggested in Scheme 2

for 1:1 stoichiometric conditions.

Kinetic Measurements. Kinetics of the electron-transfer

processes was studied by following the increase in absorbance

in the UV-vis spectrum of reduced cobalamin at 530 nm. Under

pseudo-first-order conditions with respect to NP, the electron-

transfer step occurred within the time scale 0.05-5 s, depending

on the selected experimental conditions. The kinetic traces

obtained in the pH range 1.8-6 gave best mathematical fits to

a kinetic model involving two first-order reactions. Analysis of

the data with a two-exponential function indicated that the rate

of both the slow and fast component increased on increasing

pH and nitroprusside concentration. However, at a given pH

value, the absorbance change resulting from the faster reaction

increased on increasing the NP concentration, whereas that

attributable to the slower component progressively decreased,

and could no longer be observed at higher [NP]. The concentra-

tion of NP at which the contribution of the slower component

became negligible decreased from ca. 0.06 M at pH 1.8 to ca.

Figure 8. Absorbance/time plots recorded at pH 7.4 for the reaction of

-

4

Cbl(II) (2 × 10 M) with an excess of NP. Concentrations of NP: 4 (a),

4

8

(b), 16 (c), 30 (d), 60 (e), 100 (f), and 150 × 10^-M (g).

contribution of inner-sphere electron transfer is significant under

conditions where [NP]/[Cbl(II)] e 5, whereas at higher NP

concentrations the outer-sphere pathway leading to formation

of 2, plays a dominant role (the relative contributions of both

reaction routes at a given NP concentration also depended on

pH, as discussed in more detail below).

Careful examination of the spectral changes that occur on a

long time scale (100-2000 s) and a comparison with the

respective slow spectral changes observed for equimolar con-

centrations of the reactants, enabled elucidation of the nature

of the slow reaction steps, which in the presence of ca. 10 times

0

.006 M at pH 5. At pH > 5 the contribution of the slower

(and higher) excess of sodium nitroprusside ([NP] > 0.001 M)

component to the overall absorbance change could not be

observed even at the lowest NP concentrations required to

maintain pseudo-first-order conditions (0.001 M).

occur only at pH > 5. It may be concluded from these studies

that the subsequent reaction observed in the pH range 5-7

mainly involves substitution of nitroprusside in complex 2

(

formed as the dominant initial reaction product) by cyanide,

Experimental results clearly indicated that the two processes

observed at pH e 5 occur on a relatively similar time scale,

such that the rate constants obtained for the slow and fast

components differed by a factor e 5. Consequently, although

the general reactivity trends described above were evident from

two-exponential analysis of kinetic traces, clear differentiation

between the faster and the slower component was difficult,

particularly when the amplitude attributable to one of the

components was small. This resulted in a considerable scatter

of the kobs(1) and kobs(2) values (ascribed to the faster and slower

reaction, respectively) obtained from the two-exponential analy-

sis (plots of kobs(1) and kobs(2) versus [NP] are shown in Figure

S6, Supporting Information). Thus, although the two-exponential

analysis was applied to kinetic traces that indicated dual reaction

pathways, it allowed only a crude estimation of the selected

kinetic parameters that characterize the reaction system (see text

below). On the contrary, analysis of the kinetic traces according

to a single-exponential model, although these resulted in much

as shown in reaction 7

II

+

-

[

Cbl(III)-(µ-NC)-Fe (CN) (NO )] + CN a

4

II

+ 2-

Cbl(III)CN + [Fe (CN) (NO )] (7)

5

A comparison of the stability constant reported for Cbl(III)CN

1

4

-1

(

×

1.2 × 10 M ) with that determined for complex 2 (K1 ) 5

6

-1 38

10 M ), shows that reaction 7 is indeed thermodynami-

cally favored. On further increasing pH, formation of the

R-cyano substituted species could be observed, as evidenced

by the emergence of a characteristic shoulder in the UV-vis

spectrum at ca. 600 nm (this process was also accompanied by

a decrease in absorbance at 540 nm following the initial rapid

absorbance increase, compare the kinetic traces d-g reported

in Figure 8). The contribution of this reaction path to the overall

process was only small at physiological pH, but became

significant at pH g 8. Spectral changes observed under these

1344 J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

On the basis of the results obtained from the one- and two-

exponential analysis, we ascribe the fast and slow components

to the outer- and inner-sphere electron transfer pathways,

respectively. The contribution of the inner-sphere path, however,

was small under the employed experimental conditions (par-

ticularly at pH > 4), such that the experimental points presented

in Figure 9 mainly reflect the kinetics of outer-sphere electron

transfer. Consequently, reliable values of k5K2 could only be

determined from fits to eq 8 in the pH range 1.8-3.5. These

values, however, compare relatively well with that estimated

from the plots of kobs(2) versus [NP] obtained from the two-

exponential analysis of the kinetic traces in the pH range 1.8-

5

, which are summarized in the third column of Table 1 (in

this case, the k5K2 values correspond directly to the slopes of

the plots of kobs(2) vs [NP] presented in Figure S6-b).

The k6K4 values characterizing the outer-sphere electron

transfer between 1p and nitroprusside could be determined from

the single-exponential fits in the pH range 1.8-6. The rate

constants k6 calculated from these values and from the equi-

librium constants K2 (determined as the third parameter from

the fits of experimental data to eq 8), increase significantly on

increasing pH. This effect could be directly correlated with pH,

as shown in Figure 10.

The main conclusions emerging from these kinetic studies

can be summarized as follows. Under pseudo-first-order condi-

tions with respect to NP, the contribution from inner-sphere

electron transfer to the observed redox process decreases on

increasing pH, and becomes negligible at pH > 5. Such a

reactivity trend is in line with the influence of pH on the

thermodynamics of inner-sphere electron transfer described in

the previous section. It can be expected that on increasing pH,

the equilibrium characterized by the rate constants k5 and k-5

Figure 9. Plots of kobs vs [NP] resulting from the one-exponential fit of

kinetic traces at different pH (as indicated in the figure). [Cbl(II)] ) 1 ×

-

4

1

0

M, 25 °C.

less satisfactory mathematical fits (compare Figure S7, Sup-

porting Information), did give consistent and reproducible results

and enabled determination of kinetic and thermodynamic data

which could not be obtained from the two-exponential model.

Thus, both methods of analysis of the experimental data were

applied in order to provide kinetic characterization of the

reaction system.

Figure 9 presents the plots of the observed first-order rate

constants versus [NP] obtained from the single-exponential fits

of the kinetic traces in the pH range 1.8-6. Two noteworthy

features of these plots are the nonlinear dependence of the

observed rate constants on the [NP], and the marked dependence

of the reaction rate on pH. The reactions considered to account

for the observed kinetics involve a rapid preequilibrium which

leads to the formation of the cyano-bridged precursor, followed

by the rate-determining inner- and outer-sphere electron-transfer

steps, as presented in Scheme 4. The suggested mechanism is

consistent with the rate law described by eq 8

(Scheme 4) gradually shifts to the left and thus competes less

efficiently with outer-sphere electron transfer between 1p and

NP. Despite the modest accuracy of the data characterizing the

inner-sphere path, they do provide evidence that the rate of this

process increases in the pH range 2-5. The apparent pKa value

of 3.3 determined from the pH profile constructed from the k5K2

values given in Table 1, (Figure S8, Supporting Information)

is similar to that obtained for the rate constant k6 that

characterizes the outer-sphere reaction path. The observed pH

dependences of the inner- and outer-sphere reactions can be

reasonably attributed to the acid-base equilibrium presented

in Scheme 5. Electrochemical measurements indicated that this

equilibrium significantly influences the redox properties of the

cobalt center in complex 1 (as described in more detail below).

The pKa value of 3.4 estimated for this process from the redox

potential measurements is in close agreement with that obtained

from the kinetic data. The observed ca. 100-fold acceleration

of the outer-sphere electron-transfer process resulting from

coordination of dimethylbenzimidazole to the cobalt center,

compares well with that reported in the literature for the

2

K [NP]

-

2

^kobs ) k [CN ] + k

⁺^k6

-5

5

^{1

}

{

}

+ K [NP]

1 + K [NP]

2

(

8)

Due to the reversible nature of the inner-sphere electron-transfer

path, it is essential to include the back reaction characterized

by the rate constant k-5 in the overall reaction scheme. However,

the influence of the reverse reaction path on the kinetics was

meaningful only under nearly stoichiometric concentrations of

the reactants (as described earlier), or in the presence of

externally added cyanide. In the presence of the large excess

of NP employed in the kinetic studies, its contribution was very

small (as indicated by negligible intercepts in the plots presented

39

reduction of selected organic halides by Cbl(II).

The K2 values reported in Table 1, although considerably

scattered, indicate an increase in the stability of the precursor

complex at higher pH compared to that observed in acidic

medium. This trend can be rationalized in terms of the higher

-

in Figure 9), and thus the term k-5[CN ] in eq 8 was neglected

in the subsequent treatment of the data. To resolve the overall

reaction that proceeds via parallel pathways with different

mechanisms into the contribution of each pathway, the data

presented in Figure 9 were fitted to eq 8 (solid lines in Figure

3

8b

(38) (a) This value was determined at pH 7 using a competition technique in

-

which N

3

was employed as the competing nucleophile (b) Nome, F.;

9

). The thermodynamic and kinetic parameters obtained from

Fendler, J. H. J. Chem. Soc., Dalton Trans. 1976, 1212.

(39) Blaser, H. U.; Halpern, J. J. Am. Chem. Soc. 1980, 102, 1684.

the respective fits are summarized in Table 1.

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1345

A R T I C L E S

Wolak et al.

Table 1. Kinetic and Thermodynamic Parameters for the Inner- and Outer-Sphere Electron Transfer Reaction of Cbl(II) with NP

a

-1 -1

b

-1 -1

,^aM^-²s^-¹

,^aM^-¹

k

6

,^cM^-¹s^-¹

pH

k

5

K

2

, M s

k

5

K

2

, M s

k K

6 2

K

2

4

5

2

1

.8

.5

.0

.4

.5

.0

.7

.0

.3

.8

(1.8 ( 0.4) × 10

(4.3 ( 0.4) × 10

(6.4 ( 1.3) × 10

(2.5 ( 0.1) × 10

(4.7 ( 0.3) × 10

(9.3 ( 0.2) × 10

(16.7 ( 1.4) × 10

(1.1 ( 0.3) × 10

(3.5 ( 0.7) × 10

(1.3 ( 0.3) × 10

12 ( 6

22 ( 6

51 ( 12

(9 ( 5) × 10

3

2

3

4

5

(1.6 ( 0.5) × 10

(2.5 ( 0.8) × 10

2

5

6

3

4

(8.0 ( 1.5) × 10

(2.0 ( 0.2) × 10

(4.4 ( 0.4) × 10

(1.0 ( 0.1) × 10

27 ( 4

50 ( 6

93 ( 18

(7.4 ( 1.3) × 10

(8.8 ( 1.4) × 10

(1.1 ( 2.6) × 10

2

d

(23.4 ( 0.7) × 10

(27.3 ( 2.9) × 10

6

4

e

(1.3 ( 0.3) × 10

118 ( 34

126 ( 14

(1.1 ( 0.4) × 10

(1.4 ( 0.1) × 10

(1.1 ( 0.2) × 10

a

Values determined from the fit of experimental data to eq 8. ^bValues determined from the two-exponential analysis of kinetic traces. ^cCalculated from

d

the values of k6K2 and K2 obtained from the fit of the experimental data to eq 8. Reliable determination of the k5K2 parameter was not possible (error limits

larger than the values obtained from the fit). Inner-sphere electron-transfer not observed.

e

Figure 10. Plot of the second-order rate constant, k6, versus pH for outer-

Figure 11. Comparison of the observed rate constants obtained from a

single-exponential analysis of the absorbance/time plots at pH 5.8 and 7.4.

Solid line represents the best fit of the data obtained at pH 5.8 to eq 8.

II

+ 2-

sphere electron transfer between [Cbl(II)-(µ-NC)-Fe (CN)4(NO )] and

II

+ 2-

[

Fe (CN)5(NO )]

.

Scheme 5

On increasing pH above 6, additional reactions which

contributed to the absorbance change on a stopped-flow time

scale could be observed. On the basis of the kinetic and

spectroscopic measurements, these processes were identified as

-

subsequent reactions involving the attack of CN on the

primarily reaction products formed in the electron transfer step.

This mainly includes formation of the R-cyano substituted

species (reaction path B in Scheme 2) and, to a lesser extent,

rate of NP coordination (i.e., larger k2 value) to base-on reduced

cobalamin (which involves binding of NP at the vacant

coordination site) compared to base-off Cbl(II) (where substitu-

tion of a labile water molecule by nitroprusside must occur).

The observed effect, however, is small. This observation is in

-

substitution of NP by CN in the initially formed complex 2

(reaction 7). Although these reactions occurred on a minute time

scale in the pH range 5-6, at higher pH values their rate became

close to that observed for the electron transfer steps. As the

difference between the rate of electron transfer and the

subsequent steps was still relatively large in the pH range 6-8,

the interference from the latter processes observed under these

pH conditions was quite small (although evident). Attempts were

made to separate the interfering processes from the electron

transfer steps by arbitrary choice of the time range in which

the kinetic traces could be fitted with a single-exponential

function. The resulting data presented in Figure 11 show that

the observed rate constants do not differ significantly from that

determined in the pH range 4.5-6. However, the data obtained

in the pH range 6-8 could not be satisfactorily fitted to eq 8.

2

agreement with previous findings on the coordination of NO

_a_n_d_s_e_l_e_c_t_e_d_o_r_g_a_n_i_c_r_a_d_i_c_a_l_s₄0 to the base-on and base-off forms

-

1

of Cbl(II). The equilibrium constant K4 ) 126 M determined

at pH 5.8 can be compared with that reported for the Cbl(II)CN

adduct at basic pH (reaction 9)

K₇

-

2

-1

Cbl(II) + CN {

\

} [Cbl(II)CN]

K ) 2.4 × 10 M

7

(9)

The lower equilibrium constant found in the present system is

II

+ -

in line with the expected influence of a [Fe (CN)4(NO )] group

coordinated to the cyanide carbon, which decreases the basicity

of the adjacent nitrogen and hence weakens the Co-NC bond.

The one-exponential analysis could not be applied to the

kinetic traces obtained at pH > 8, which gave good mathemati-

cal fits only for a model involving a three-exponential function

(a typical kinetic trace obtained at pH 8.5 is presented in Figure

(40) Bakac, A.; Espenson, J. H. Inorg. Chem. 1989, 28, 4319.

1

346 J. AM. CHEM. SOC. VOL. 125, NO. 5, 2003

9

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

-

3

-3

Figure 12. Differential pulse voltammograms of (a) aquacobalamin (1 × 10 M, pH 7.0), (b) NP (1 × 10 M, pH 7.4), (c) complex 2 formed by mixing

-

3

-3

of aquacobalamin (1 × 10 M) with NP (1 × 10 M) at pH 7.4, (d) comparison of differential pulse voltammograms recorded for complex 2 at pH ) 7.4

solid line) and 2.5 (dotted line). Symbols ICo, IICo, IFe, and IIFe denote peaks corresponding to the first and second reduction of the cobalt and iron center,

respectively. I′Co, II′Co, I′Fe, and II′Fe represent the corresponding peaks observed at low pH. Experimental conditions: DPV recorded in 0.1 M NaClO4, pH

7.4 and 2.5 adjusted with NaOH and HClO4, respectively; scan rate 10 mV/s, pulse height 25 mV, step duration 0.2 s.

(

)

S9, Supporting Information). However, due to the fact that the

observed processes occurred on a similar time scale (and in

addition, small amplitudes attributable to the fastest and slowest

component were observed), such analysis did not yield the rate

constants which would enable reliable and reproducible analysis

of the kinetic data. Nevertheless, it could be concluded

qualitatively that the electron-transfer processes also in this case

occurred on a time scale similar to that observed in the pH range

change in oxidation states are observed at ca. -0.01 and -0.86

V in the DPV of aquacobalamin, whereas those observed for

II

+ 2-

the first and second reduction process in [Fe (CN)5(NO )]

appear at -0.36 and -0.58 V, respectively. The observed peaks

correspond to the oxidation-reduction processes for which the

1

III

II

2

II

I

potentials E (Co /Co ) ) -0.02 V, E (Co /Co ) ) -0.83V,

1

-

2

-

2-

E (NP/NP ) -0.34 V and E (NP /NP ) -0.58 V (vs Ag/AgCl)

were reported in the literature.

1

8,34,42

Stepwise addition of NP

4

.5-8.

to the aquacobalamin solution resulted in the disappearance of

III

II

Electrochemical Studies. Due to the fact that complex 1

the Co /Co peak at -0.01 V (peak ICo in Figure 12a). This

was accompanied by a progressive broadening and a shift of

the second reduction peak of cobalamin to a more negative

potential. In a 1:1 molar mixture of the reactants, this peak is

formed in the reaction of reduced cobalamin with NP repre-

sents a one-electron reduced form of [Cbl(III)-(µ-NC)-

Fe (CN)4(NO )] (2), electrochemical techniques provide an

opportunity to investigate the electronic properties of the metal

centers in 1 and 2 and to compare them with that observed for

the respective mononuclear species. Figures 12a-c show the

differential pulse voltammograms of aquacobalamin, sodium

nitroprusside and an equimolar mixture of both species (i.e., of

_c_o_m_p_l_e_x₂₎_a_t_p_H₇_.₄_.₄1 Reduction peaks of mononuclear

cobalamin species corresponding to the Co /Co and Co /Co

II

+ -

(

41) Differential pulse voltammetry offers a better resolution of poorly separated

redox waves compared to cyclic voltammetry, and for that reason is mainly

used in the discussion of the reductive processes observed for compex 2.

Cyclic voltammograms recorded in the corresponding potential range

(compare Figure S10, Supporting Information) provided additional informa-

tion on the reoxidation processes from reverse anodic scans. Detailed

discussion of these data, however, is beyond the scope of the present study.

42) Carapu c¸ a, H. M.; Simao, J. E. J.; Fogg, A. G. J. Electroanal. Chem. 1998,

455, 93, and references therein.

(

III

II

I

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1347

A R T I C L E S

Wolak et al.

Scheme 6

located at -1.0 V (peak (I + II)Co in Figure 12c), and

corresponds to an irreversible two-electron reduction wave with

a maximum at ca. -1.04 V in the cyclic voltammogram

recorded under the same experimental conditions (Figure S10-

a, Supporting Information). In addition, three new peaks appear

at -0.19, -0.36, and -0.58 V, the latter two located at

potentials very similar to that characteristic for the first and

second reduction of NP at pH 7.4. The same final differential

pulse voltammogram resulted from the stepwise addition of one

mole equivalent of aquacobalamin to the NP solution, where

in this case splitting of the first reduction peak of NP into two

peaks located at - 0.19 and - 0.36 V was accompanied by

appearance of a new peak at -1.0 V.

In contrast to the large effect on the redox properties of the

cobalt center, the redox behavior of nitroprusside does not

change significantly upon coordination to aquacobalamin. This

is evidenced by the similar location of the peaks attributable to

the iron center in the oligonuclear complex compared to that

observed for [Fe (CN)5(NO )] . Splitting of the peak char-

acteristic for the one-electron reduction of NP (peak IFe in Figure

12b) into two peaks centered at -0.19 and -0.36 V possibly

reflects formation of cis and trans isomers of 2, i.e., [Cbl(III)-

II

+ 2-

II

+ -

(µ-NCax)-Fe (CNax)(CNeq)3(NOeq )] and [Cbl(III)-(µ-NCax)-

II

+ -

Fe (CNeq)4(NOax )] , respectively. Electrochemical reduc-

tion of the trans isomer is expected to result in its rapid

and irreversible decomposition to cyanocobalamin and

I

+ 2-

We interpret the observed changes in the DPV of aqua-

[Fe (CNeq)4(NO )] . A positive shift in the one-electron

reduction potential of the iron center compared to that of free

III

II

cobalamin in terms of a strong negative shift of the Co /Co

redox potential to values close to that observed for a Co /Co

II

I

2-

[Fe(CN)5NO] , as a result of a fast irreversible reaction

reduction, as a result of substitution of a water molecule in

following the electron-transfer step, can therefore be expected

for the trans isomer. The cis isomer, however, does not undergo

decomposition upon one-electron reduction, but is expected to

release cyanide in a manner similar to that observed for free

+

[

Cbl(III)H2O] by the bridging cyanide. Consequently, reduction

III

II

of the cobalt center in 2 consists of the consecutive Co /Co

and Co /Co electron transfer steps that occur at nearly the same

potential, and is observed as a two-electron reduction wave at

II

I

1

-

NP and, consequently, may exhibit a similar E (NP/NP ) value.

Thus, it seems reasonable to ascribe the two peaks at -0.19

and -0.36 V to the trans- and cis-isomeric forms of 2,

respectively. The position of the second reduction potential of

NP in the oligonuclear complex is very similar to that reported

-

1.04 V in the respective cyclic voltammogram (the irrevers-

ibility of this wave results from the occurrence of chemical

reactions upon formation of the tetracoordinate Cbl(I), compare

Scheme 6). Similar changes in the redox properties of aqua-

cobalamin resulted from coordination of [Fe(CN)6]⁴to aqua-

cobalamin (Figure S11, Supporting Information), where the

-

II

+ 2-

for [Fe (CN)5(NO )] . This suggests that similar mechanisms

4

are operating in the second reduction process for the bound

and free nitroprusside.

II

3-

43

[

Cbl(III)-(µ-NC)-Fe (CN)5]

complex is formed. The

III

II

observed large negative shift in the Co /Co redox potential is

comparable with that resulting from coordination of CN to

Positive shifts in the potentials attributed to both metal centers

in 2 were observed in the differential pulse voltammograms on

-

III

aquacobalamin (in this case, a very stable C-bonded Co -CN

_c_o_m_p_l_e_x_i_s_f_o_r_m_e_d₎_.₁0

(44) Detailed electrochemical studies reported in the literature indicate that the

nature of the reductive process occurring at the second reduction potential

of NP is complicated and, in addition to a reversible one-electron reduction

2

-

(

43) Due to the absence of the redox-active NO ligand, cyclic and differential

of [Fe(CN)

4

NO] , may involve its multielectron reduction, as well as

42

II

3-

2-

pulse voltammograms of [Cbl(III)-(µ-NC)-Fe (CN)

5

]

are simpler than

comproportionation and adsorption phenomena. However, for the sake

II

+

2-

those obtained for [Cbl(III)-(µ-NC)-Fe (CN)

4

(NO )] . This facilitates

4

of clarity, only the one-electron reduction of [Fe(CN) NO] (which is

observation of changes in the electrochemical properties of the metal centres

resulting from the formation of cyano-bridged species.

expected as the predominant process occurring at the second reduction

potential under our conditions) is included in the discussion.

1348 J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

The less marked positive shifts observedfor peaks IFe and

IIFe (from -0.36 to -0.25 V and from -0.58 to -0.46 V,

respectively, compare Figure 12d) are similar to those reported

II

+

2-

45

for the monomeric [Fe (CN)5(NO )] complex. On the basis

of literature data on the redox behavior of sodium nitro-

prusside,

3

4,42

as well as that reported for the reduction of

cyanocobalamin (in which the cobalt center undergoes a

simultaneous two-electron reduction similar to that observed

1

0

in the system presently studied ), the differential pulse and

cyclic voltammograms obtained at high (pH > 6) and low

(pH < 3) pH, can be accounted for by the reaction sequences

A and B (for high and low pH, respectively) presented in

Scheme 6.

Cyclic and differential pulse voltammograms recorded for

equimolar mixtures of reduced cobalamin and NP at pH 2 were

similar to those obtained for complex 2 under similar experi-

mental conditions, except that the peak at -0.19 V (attributed

Figure 13. Plot of the potential observed for reduction of the cobalt center

in complex 2 (peak (I + II)Co) versus pH.

lowering the pH from 6 to 2. The most significant changes

II

to the one-electron reduction of the Fe center in the trans isomer

III

I

involved the Co /Co potential, as evidenced by the gradual

of 2) was no longer observed. The similarity of cyclic and

differential pulse voltammograms of complexes 1 and 2 provides

confirmatory evidence that the stable product formed in the

positive shift of the (I + II)Co reduction peak from -1.0 to

-

0.7 V. This effect can be clearly seen from Figure 12d, which

shows a comparison of the differential pulse voltammograms

recorded at pH 7.4 and 2.5. The pH dependence of the Co /

Co potential presented in Figure 13 clearly shows that the

II

+ 2-

reaction of Cbl(II) with [Fe (CN)5(NO) ] in the low pH range

III

is in fact a one-electron reduced form of 2.

I

An important conclusion that arises from the above outlined

observed shift reflects the pH dependent process for which the

pKa ) 3.4. This value is very close to that determined kinetically

for the base-on/base-off equilibrium presented in Scheme 5, and

provides confirmatory evidence for our earlier conclusions

concerning the influence of this equilibrium on the rate of inner-

and outer-sphere electron transfer.

II

+ 2-

observations is that coordination of [Fe (CN)5(NO )] to

cobalamin shifts the redox potential of the Co(III) center to more

negative values than observed for the first (and second) reduction

of bound NP. This means that the reaction of Cbl(II) with NP

II

must involve formation of the Co -(µ-NC)-Fe intermediate

-

4

-4

Figure 14. (a) Release of NO after addition of Cbl(II) (2 × 10 M) to NP solution (2 × 10 M) at pH 7.4 (0.1 M Tris-HClO4), (b) as in (a), [NP] ) 1

-

2

2+

×

10 M, (c) decay of NO signal after addition of aqueous NO solution to deoxygenated buffer (pH 7.4), (d) as in (c), after addition of [Fe(H2O)6] (5

-

6

10 M).

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1349

A R T I C L E S

Wolak et al.

Scheme 7

which undergoes electron transfer to form a highly thermody-

Our observation that NO is formed as a side product in the

observed redox process is not unexpected in view of the

literature data on the reactivity of nitroprusside, which is known

III

I

namically favored Co -(µ-NC)-Fe electronic isomer.

Release of NO at Physiological pH. Concentrations of NO

released in the reaction of reduced cobalamin with NP at

physiological pH was monitored with the use of an NO selective

-

to release CN rather than NO in the first reaction step following

2

4-27,42

the one-electron reduction process.

However, release of

-

4

-4

electrode at different Cbl(II) (1 × 10 - 5 × 10 M) and NP

NO may also occur as a side reaction that leads to stabilization

of the reduced iron center, and may thus account for the

observed small initial production of NO. This and other

processes which are suggested to account for the observed

patterns of NO production following the inner- and outer-sphere

electron-transfer reactions are summarized in the reaction

sequences A and B of Scheme 7. According to this scheme,

the second stage of NO release that occurs on a long time scale

-4

-2

concentrations (1 × 10 - 1 × 10 M). A characteristic two-

stage pattern of NO production observed after addition of Cbl(II)

to a NP solution under oxygen-free conditions is shown in

Figure 14, which reports the changes in [NO] after mixing

Cbl(II) with an equimolar concentration (a) and with a large

excess of NP (b) (i.e., under the conditions where the inner-

and outer-sphere electron-transfer reactions are the dominating

reaction paths, respectively). It becomes evident from these data

that release of NO is rather slow, and the measured NO

concentrations are low (the maximal observed NO concentra-

tions ≈ 2% of that expected for stoichiometric release of NO

from one-electron reduced NP). Correlation of the release of

NO with the UV-vis spectral changes recorded under the same

experimental conditions clearly indicated that the maximal

increase in [NO] during the second, slower stage of NO release

I

+ 2-

results from slow decomposition of [Fe (CN)4(NO )] . It

should be noted that due to the high affinity of the Co(III) center

-

of cobalamin for CN , the electron transfer and subsequent

substitution processes observed in the present system at pH 7.4

can be summarized by the overall reaction 10

II

+ 2-

Cbl(II) + [Fe (CN) (NO )]

f

5

I

+ 2-

Cbl(III)CN + [Fe (CN) (NO )] (10)

4

(observed ca. 50 min after mixing of the reactants, independent

of the [NP] employed) occurs on a much longer time scale than

the electron-transfer processes. The first stage of NO release

could, however, be roughly correlated with the rate of electron

transfer. In addition, experiments performed at different Cbl(II)

and NP concentrations showed that the amount of NO released

was proportional to the concentration of the reductant, but did

not depend on the concentration of NP.

I

+ 2-

Thus, [Fe (CN)4(NO )] is the main iron complex formed

after both the inner- and outer-sphere electron-transfer reactions.

Literature data provide evidence that this ion undergoes further

decomposition to NO and various iron(II) complexes such as

3

-

4-

2+

[

Fe(CN)5H2O] , [Fe(CN)6] , and [Fe(H2O)6] , from which

the latter two were suggested as the ultimate reaction prod-

2

5,26

ucts.

The NO concentrations measured in our experiments

are, however, significantly lower than that expected for the

stoichiometric release of NO from the [Fe (CN)4(NO )] ion.

(

45) No significant dependence on pH was observed for the peak ascribed to

I

+ 2-

the one-electron reduction of the trans isomer of 2 in the studied pH range.

1350 J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003

Interaction of Vitamin B12r with Nitroprusside

A R T I C L E S

This observation can be accounted for by: (i) only partial

decomposition of the tetracyanonitrosylferrate ion on the time

scale of our measurements⁴⁶(only scarce literature data could

be found on the kinetics of this process); (ii) scavenging of NO

by the iron(II) complexes formed during decomposition of

cyanide ligand from the coordinated iron complex, and (ii)

dechelation of the DMBI base from the cobalt center. A strong

influence of pH on the subsequent substitution processes leading

to the formation of the final reaction products Cbl(III)CN and

[Fe (CN)4(NO )] , reflects the competition between H and

the Co(III) center for the cyanide released during the electron-

transfer step.

I

+

2-

+

I

+ 2-

[Fe (CN)4(NO )] . The latter possibility is supported by the

characteristic rapid decay of NO following its maximum

production. This feature was particularly evident in the experi-

ments involving excess of NP, Figure 14b, and may result from

reactions such as that given in (11), for which the equilibrium

With respect to the possible influence of cobalamin on the

physiological activity of NP, the following points should be

addressed. The electrochemical results indicate that formation

3

-1

II

+ -

constant KNO ) 1.2 × 10 M (pH 5.0, 23 °C) has been

reported

of [Cbl(III)-(µ-NC)-Fe (CN)4(NO )] (2) does not signifi-

cantly affect the redox properties of nitroprusside. Thus,

biological processes involving reductive activation of nitro-

prusside to initiate the release of NO should not be significantly

influenced by formation of 2 in vivo. Experimental results also

indicated that substitution of NP in complex 2 by cyanide is

highly thermodynamically favored. This result is in agreement

with the significantly higher stability constant of Cbl(III)CN

compared to that determined for complex 2. Thus, although

4

7

2

+

2+

[

Fe(H O) ] + NO a [Fe(H O) NO] + H O (11)

2 6 2 5 2

A similar effect was indeed observed upon addition of

+

[

Fe(H2O)6]²to an aqueous NO solution, which resulted in a

significantly faster decay of the NO signal compared to that

observed for a blank NO solution of the same concentration, as

reported in Figures 14c and d. Thus, binding of NO to Fe(II)

+

binding of NP to [Cbl(III)H2O] is kinetically faster than the

-

corresponding reaction with CN , and in the presence of both

I

2-

-

complexes formed during decomposition of [Fe (CN)4NO]

CN and NP would prevail on a shorter time scale, the

may in fact lower the NO concentrations measured by the NO-

electrode in the experiments involving Cbl(II) and NP.

Taking into account that the affinity of many physiological

thermodynamically driven conversion of 2 to Cbl(III)CN and

NP will occur on a longer time. Results obtained at physiological

pH indicate that the one-electron reduction of nitroprusside in

2+

-

targets for NO is significantly higher than that of [Fe(H2O)6] ,

complex 2 leads to the release of CN , which at this pH

formation of [Fe(H2O)5NO]²should not affect the physiological

+

I

+ 2-

effectively substitutes the [Fe (CN)4(NO )] group in the

+

activity of NP. Nevertheless, due to the fact that the NO

cyano-bridged complex 1 to form Cbl(III)CN and free

I

+ 2-

character of the nitrosyl ligand (and therefore its pre-

disposition to undergo nucleophilic attack) is retained in

[Fe (CN)4(NO )] . The latter species, however, is expected to

undergo the same metabolic transformations leading to donation

of NO as that occurring after the one-electron reduction of free

NP in vivo. It may, therefore, be concluded that as far as the

results of the present study are concerned, binding of NP to

cobalamin should neither significantly affect the NO-donating

ability of NP, nor exclude cobalamin as an effective scavenger

of cyanide released from metabolized NP.

I

+ 2- 7,27

[

Fe (CN)4(NO )]

,

interaction of one-electron reduced

nitroprusside with cellular nucleophiles rather than spontaneous

release of NO appears a more probable metabolic route leading

to the observed pharmacological effects.

Conclusions

The results of this study clearly demonstrate that different

reaction paths are followed by the reactants depending on the

experimental conditions employed. The most important param-

eter in this respect is pH because this affects the rate and

thermodynamics of the observed inner- and outer-sphere

electron-transfer processes through the pH-dependent equi-

libria involving the precursor complex, [Cbl(II)-(µ-NC)-

Acknowledgment. Studies at UEN were supported by the

Deutsche Forschungsgemeinschaft as part of SFB 583 “Redox-

Active Metal Complexes: Control of Reactivity via Molecular

Architecture”. Studies at UJ were supported by the State

Committee for Scientific Research, Poland, KBN (3T09A13518).

M.W. gratefully acknowledges support from DAAD.

Supporting Information Available: Figures S1-S11 referred

to in the text are available via the Internet at http://pubs.acs.org.

See any current masthead page for ordering information and

Web access instructions.

II

+ 2-

Fe (CN)4(NO )] . These include (i) the release of a peripheral

(

46) Concentrations of nitric oxide were monitored for ca. 2 h after mixing of

the reactants.

(47) Wanat, A.; Schneppensieper, T.; Stochel, G.; van Eldik, R.; Bill, E.;

Wieghardt, K. Inorg. Chem. 2002, 41, 4.

JA0210852

J. AM. CHEM. SOC.

9

VOL. 125, NO. 5, 2003 1351

Article Doi

Mechanistic studies on the interaction of reduced cobalamin (vitamin B12r) with nitroprusside

DOI: 10.1021/ja0210852

Source and publish data:

Authors:

Article abstract of DOI:10.1021/ja0210852

Full text of DOI:10.1021/ja0210852

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