Spectral and electronic properties of nitrosylcobalamin

Article

pubs.acs.org/IC

Spectral and Electronic Properties of Nitrosylcobalamin

Ivan G. Pallares and Thomas C. Brunold*

Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States

*

S Supporting Information

ABSTRACT: Nitrosylcobalamin (NOCbl) is readily formed when Co(II)-

balamin reacts with nitric oxide (NO) gas. NOCbl has been implicated in the

inhibition of various B -dependent enzymes, as well as in the modulation of

1

2

blood pressure and of the immunological response. Previous studies revealed

that among the known biologically relevant cobalamin species, NOCbl

possesses the longest bond between the Co ion and the axially bound 5,6-

dimethylbenzimidazole base, which was postulated to result from a strong trans

inﬂuence exerted by the NO ligand. In this study, various spectroscopic

(

electronic absorption, circular dichroism, magnetic circular dichroism, and

resonance Raman) and computational (density functional theory (DFT) and

time-dependent DFT) techniques were used to generate experimentally

validated electronic structure descriptions for the “base-on” and “base-oﬀ”

forms of NOCbl. Further insights into the principal Co−ligand bonding

interactions were obtained by carrying out natural bond orbital analyses. Collectively, our results indicate that the formally

unoccupied Co 3d orbital engages in a highly covalent bonding interaction with the ﬁlled NO π* orbital and that the Co−NO

bond is strengthened further by sizable π-backbonding interactions that are not present in any other Co(III)Cbl characterized to

2

z

−

date. Because of the substantial NO to Co(III) charge donation, NOCbl is best described as a hybrid of Co(III)−NO and

•

Co(II)−NO resonance structures. In contrast, our analogous computational characterization of a related species,

−

superoxocobalamin, reveals that in this case a Co(III)−O description is adequate due to the larger oxidizing power of O₂

2

versus NO. The implications of our results with respect to the unusual structural features and thermochromism of NOCbl and

the proposed inhibition mechanisms of B -dependent enzymes by NOCbl are discussed.

1

2

7

,8

INTRODUCTION

oxide (NO). While these enzymes are chemically unreactive

■

9

toward NO in their resting states, the Co(II)Cbl intermediates

Cobalamins (Figure 1) consist of a six-coordinate low-spin

Co(III) ion that is ligated equatorially by the four nitrogens of a

tetrapyrrole macrocycle, known as the corrin ring. The “lower”

axial position is occupied by a nitrogen from the 5,6-

dimethylbenzimidazole (DMB) base that is part of an

intramolecular nucleotide loop bound to the corrin ring at

C . At low pH, the coordinating nitrogen of the DMB group

becomes protonated, which converts the cobalamins from their

base-on” state to their “base-oﬀ” form in which a solvent-

derived water molecule now serves as the lower ligand. An

excellent model of these base-oﬀ species at neutral pH is

provided by cobinamides, which are naturally occurring

formed during catalysis (in the case of MMCM) or cofactor

reactivation (MetH) are believed to be susceptible to reactions

with this neutral radical species. In support of this assumption,

in vitro studies have indicated that NO reacts with Co(II)Cbl

on a microsecond time scale to yield nitrosylcobalamin

1

0,11

(

NOCbl),

while in vivo studies in animals revealed that

17

hydroxycobalamin supplementation can inhibit the physiolog-

12

ical response to NO, due to eﬃcient NO scavenging by the

resulting Co(II)Cbl formed in the cell. Because NOCbl does

not support the catalytic activities of MetH and MMCM, high

cellular levels of NO are expected to result in a buildup of

homocysteine and methylmalonyl-CoA, thereby causing

disruption of the homocysteine metabolism and, possibly, the

“

2

3

cobalamin precursors that lack the terminal DMB group.

The biologically active forms of cobalamin diﬀer with respect

to the identity of the variable upper axial ligand, with the best

characterized forms being methylcobalamin (MeCbl) and

6

induction of methylmalonic aciduria.

Despite the wealth of experimental evidence supporting the

formation of NOCbl in vivo, the electronic structure of this

species remains incompletely understood, in part because NO

is a redox-active ligand, thus making an oxidation state

4

adenosylcobalamin (AdoCbl). These molecules feature an

organometallic bond between the Co(III) ion and either a

methyl group or an ATP-derived 5′-deoxyadenosyl group. In

humans, MeCbl serves as the cofactor of methionine synthase

13

assignment for Co ambiguous₁. Using the Enemark−Feltham

4

formalism for metal nitrosyls, NOCbl can be described as a

(

MetH), involved in the synthesis of methionine from

8

5

{M−NO} type species, where the metal−NO fragment is

homocysteine, while AdoCbl is required by methylmalonyl-

CoA mutase (MMCM) for the isomerization of methylma-

6

lonyl-CoA to succinyl-CoA. Experimental studies have

Received: April 27, 2014

revealed that both MetH and MMCM are inhibited by nitric

©

XXXX American Chemical Society

A

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NMR studies by Hassanin et al. led to the suggestion that at

neutral pH, ∼33% of NOCbl is present in the base-oﬀ form,

2

with the remaining ∼67% being in the base-on form.

To improve the current understanding of the electronic

structures of NOCbl in its base-on and base-oﬀ forms, we

carried out Abs, circular dichroism (CD), magnetic CD

(

MCD), and resonance Raman (rR) spectroscopic studies of

+

NOCbl and nitrosylcobinamide (NOCbi ). The spectroscopic

data were analyzed within the framework of density functional

theory (DFT) and time-dependent-DFT (TDDFT) calcula-

tions, employed previously with great success in computational

19−21

studies of other Co(III)Cbl and Co(III)Cbi species.

To

identify the principal Co−ligand bonding interactions, we also

carried out natural bond orbital (NBO) analyses. Collectively,

our results provide signiﬁcant new insights into the spectral and

+

electronic properties of NOCbl and NOCbi . Additionally, by

carrying out an analogous computational characterization of

superoxocobalamin (O Cbl), intriguing electronic structure

2

9

diﬀerences between this {Co−O } species and NOCbl were

2

identiﬁed.

METHODS

■

(

+

Synthesis. Aquacobalamin (H OCbl ), dicyanocobinamide

2

(CN) Cbi), sodium borohydride (NaBH ), potassium formate

2

4

Figure 1. Chemical structure of nitrosylcobalamin (NOCbl), along

with the numbering scheme used for the atoms in the corrin ring.

Colored in red is the pendant 5,6-dimethylbenzimidazole (DMB)

(HCOOK), sodium nitrite (NaNO ), ascorbic acid, and copper

2

tetrachloride (CuCl ) were purchased from Sigma-Aldrich and used as

4

obtained. Gaseous nitric oxide (NO) was generated by the reaction of

+

group, which is absent in nitrosylcobinamide (NOCbi ).

NaNO with ascorbic acid and aqueous Cu(II) chloride under an

2

argon (Ar) atmosphere. NOCbl was prepared by chemical reduction

+

of ∼2 mM H OCbl with a saturated solution of HCOOK under

anaerobic conditions to yield Co(II)Cbl, which was subsequently

exposed to freshly prepared NO gas for 2 h. To halt the reaction, the

2

treated as a single unit and is characterized by the total number

of metal d and NO π* electrons. From a comparison to {M−

8

15

NO} metalloporphyrin species, the Co−NO fragment of

NOCbl is expected to adopt a bent geometry, with the NO

ligand exerting a strong trans inﬂuence. Indeed, the crystal

structure of NOCbl obtained at 100 K shows a very long Co−

N(DMB) bond (2.32−2.35 Å), a short Co−NO bond (1.91−

+

solution vials were purged with Ar. NOCbi was prepared according to

the same procedure, except that in this case ∼2 mM diaquocobinamide

2

+

2+

(

(H O) Cbi ) was used as a precursor. (H O) Cbi was synthesized

2

by the addition of NaBH to an aqueous solution of (CN) Cbi, as

4

2

3,24

described in previous reports.

The pH of the sample solutions was

1

6

7

unless otherwise indicated.

A comparison of the electronic Abs spectra of the resulting species

1

.94 Å), and a Co−N−O bond angle of ∼120°. These axial

bond distances are consistent with structures reported for

+

to published spectra for NOCbl and NOCbi conﬁrmed that the

Co(III)Cbl species featuring a strongly σ-donating upper

2

5

−

17

reactions went to completion (see Supporting Information Figure S1

for complete details). An electron paramagnetic resonance (EPR)

characterization of these samples revealed that less than 3% of

Co(II)Cbl remained in solution after NO exposure (see Supporting

Information, Figure S2). Up to 60% (v/v) degassed glycerol was added

under anaerobic conditions to all samples used for low-temperature

Abs and MCD experiments to ensure glass formation upon freezing.

Isotopically enriched samples for rR experiments were prepared by

ligand, thus supporting a Co(III)−NO description. Addi-

tionally, the visible region of the electronic absorption (Abs)

spectrum of NOCbl in aqueous solution at neutral pH is

dominated by a broad asymmetric feature centered at ∼480

11

nm, which is characteristic of Co(III)Cbl species.

The dominant contributors to this Abs feature of Co(III)Cbl

species are the so-called α/β bands that have been attributed to

an electronic transition between corrin π/π*-based molecular

1

5

the methods described above, except with the use of N-labeled

4

NaNO (99% pure, Cambridge Isotope Laboratories, Inc.). Frozen

2

orbitals. The energies of these bands have been shown to be

pellets were prepared by injecting small aliquots of ﬂuid sample into a

sensitive to the electron-donating properties of the axial

4

liquid N bath under an argon atmosphere. Additional samples for rR

2

ligands. Notably, in the case of NOCbl, the peak position of

studies were prepared by the addition of HCl to NOCbl to reach a

the α/β bands (∼480 nm) is similar to that of base-oﬀ

alkylcobalamin species in which the lower axial DMB ligand is

ﬁnal pH value of <2. The room-temperature Abs spectra of this low-

+

pH NOCbl species and NOCbi were found to be superimposable,

replaced by a water molecule (e.g., λ ≈ 460 nm for MeCbl at

indicating that the former species was cleanly converted to its base-oﬀ

max

1

8

pH ≤ 2 ). These observations suggest that the strongly σ-

donating NO ligand could promote complete dissociation of

form, as expected on the basis of the pK value of 5.1 reported for the

a

−

2

ligating DMB nitrogen of NOCbl.

Spectroscopy. Room-temperature CD and low-temperature Abs,

CD, and MCD spectra were acquired using a Jasco J-715

spectropolarimeter in conjunction with an Oxford Instruments

SpectroMag-4000 8T magnetocryostat. All MCD spectra were

obtained by taking the diﬀerence between spectra collected with the

magnetic ﬁeld aligned parallel and antiparallel to the light propagation

axis to remove contributions from the natural CD and glass strain.

Room-temperature Abs spectra were obtained with a Cary 5E UV/vis

spectrophotometer.

the DMB group under physiological conditions. However,

detailed pH titration and NMR studies by van Eldik and co-

workers led to the identiﬁcation of a pK value of 5.1 for the

a

protonation and consequent dissociation of the DMB nitrogen,

11

indicating that base-on NOCbl is favored at neutral pH. Yet,

this pK value is only ∼0.5 pH units lower than that of the free

a

1

nucleotide base (pK of 5.56 in aqueous solution ), suggesting

a

that the Co−N(DMB) bond of NOCbl is very weak. Additional

B

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4

0,42

The rR spectra were obtained upon excitation at 488.0 nm with a

Coherent I-305 Ar ion laser and ∼40 mW of laser power at the

sample. The scattered light was collected using a 135° backscattering

arrangement, dispersed by an Acton Research triple monochromator

equipped with 300, 1200, and 2400 grooves/mm gratings), and

(RI) approximation in conjunction with the SV/J

auxiliary basis

+

were employed in the evaluation of the Coulomb term for all

calculations. The TDDFT results were used as the basis for simulating

Abs and CD spectra, assuming that each electronic transition gives rise

to a Gaussian-shaped band with a full width at half maximum (fwhm)

(

−1

analyzed with a Princeton Instrument Spec X: 100BR deep depletion,

back-thinned CCD camera. Spectra were accumulated at 77 K by

placing frozen pellets into a quartz ﬁnger dewar ﬁlled with liquid N₂.

Spectra of ﬂuid solution samples, contained in sealed EPR tubes under

an argon atmosphere, were obtained with the same setup but by ﬁlling

the ﬁnger dewar with an ice/water mixture. No spectroscopic changes

attributable to photolytic or chemical degradation were observed

during data collection. All rR spectra were baseline corrected using a

piecewise line function to remove the broad nonresonant ﬂuorescence

contributions, and the intensities were normalized with respect to the

of v₁_/₂= 1250 cm . All calculated spectra were uniformly red-shifted

−1

by 2200 cm to facilitate a visual comparison with the experimental

data. Finally, oﬀ-resonance Raman spectra for the fully optimized

truncated models were computed with ORCA 2.8, by evaluating the

numerical frequencies and electronic polarizabilities of all normal

modes using DFT and the basis sets and functionals described above.

No scaling factor was applied to the computed frequencies.

The ground-state wave functions of the fully optimized models were

43

analyzed further within the natural bond orbital (NBO) framework

to assess the major bonding interactions, using the NBO5 interface as

implemented in ADF via the gennbo and adfnbo programs. The

−1

features in the region between 1100 and 1400 cm . Peak positions

−1

31

were calibrated against the 984 cm peak of a potassium sulfate

VWN/LDA and the PBE gradient corrections for exchange and

−1

32,33

standard as well as the ice peak at 228 cm for frozen samples or the

correlation,

along with the TZP basis set (without frozen core

−1

29

water feature at 1637 cm for ﬂuid solution samples.

approximation), and an integration constant of 5.0 were used to

compute the ground-state electron density. Default parameters were

chosen for the NBO5 interface. Isosurface plots of all orbitals and

electron density diﬀerence maps were generated with Pymol using

isodensity values of 0.03 au and 0.003 au, respectively.

Computational Studies. Initial coordinates for the model of

NOCbl were obtained from the highest-resolution X-ray crystal

16

structure reported by Hassanin et al. in 2010. These coordinates

+

were also used as the basis for generating an initial model of NOCbi ,

whereby the nucleotide loop was replaced by an H atom at the

phosphate position and a water molecule was placed in the lower axial

coordination site originally occupied by the DMB base (see Figure 1).

RESULTS

■

Coordinates for the O Cbl model were obtained from the structure

2

I. Abs, CD, and MCD Data. The low-temperature (4.5 K)

Abs spectrum of NOCbl in frozen solution exhibits partially

resolved bands in the lower energy region with an intensity

maximum near 20 000 cm , corresponding to the so-called α/

β bands, along with a series of broad and more intense bands

around 31 000 cm , historically referred to as the γ region

(Figure 2, top). This spectrum is very similar to those exhibited

by alkyl-Co(III)Cbl species, such as MeCbl and AdoCbl, which

26

reported by Hohenester et al. and used as is. Because of the large

number of atoms present in these species, smaller models were

prepared for TDDFT excited-state and DFT frequency calculations by

removal of atoms that were not expected to contribute to the

spectroscopic features observed experimentally. Speciﬁcally, the corrin

ring substituents were replaced by hydrogen atoms, except for the four

methyl groups at the C , C , C , and C positions (see Figure 1 for

−

1

−1

1

2

5

15

the atom numbering scheme used). Additionally, the two propiona-

mide groups at C and C , along with the nucleotide loop at C in the

2

18

7

case of NOCbl, were replaced by methyl groups. This truncation

scheme was adopted because the methyl groups at the C and C

5

15

positions were shown to be crucial for an accurate treatment of the

27

vibrational modes of the corrin ring, while the others were found to

play a role in preventing excessive ﬂattening of the corrin ring (vide

infra). To further increase the likelihood of obtaining realistic corrin

fold angles, the entire DMB base was included in all NOCbl models.

Full geometry optimizations of the complete and truncated cofactor

models were performed with the Amsterdam Density Functional

2

8−30

(

ADF) 2012 suite of programs

using the Vosko−Wilk−Nusair

31

VWN) local-density approximation (LDA) and the Perdew, Burke,

and Ernzerhof (PBE) gradient corrections for exchange and

3

2,33

correlation.

basis set

In each case, the triple ζ with polarization (TZP)

was chosen, along with an integration constant of 5.0 and

2

9,30

a small frozen core through 1s for C, N, and O and through 2p for Co

and P. The optimized models were subjected to frequency calculations

to verify that a true energy minimum was found. In each case, only

positive frequencies were obtained.

For the computation of spectroscopic properties, the ORCA 2.8

3

4

software package developed by Dr. F. Neese was employed. While

the optimized truncated models were used as-is, the full models were

modiﬁed using the truncation scheme outlined above and subjected to

partial geometry optimizations, whereby only the added H atoms were

allowed to move. Electronic transition energies and Abs intensities

3

5−37

were computed with the TDDFT method

within the Tamm−

and using the PBE functional for exchange

The Ahlrichs polarized split valence (SVP) basis

3

8,39

Dancoﬀ approximation

32,33

and correlation.

40

set was employed for all atoms, except for cobalt, the ligating

nitrogens, and the oxygen from NO, for which the triple-ζ valence

41

polarized (TZVP) basis was utilized. In each case, 40 excited states

were computed by including all one-electron excitations among

molecular orbitals within ±3 hartree of the highest occupied molecular

orbital/lowest unoccupied molecular orbital (HOMO/LUMO) gap.

To increase computational eﬃciency, the resolution of the identity

Figure 2. Abs (top), CD (center), and 7 T MCD (bottom) spectra at

various temperatures of NOCbl.

C

dx.doi.org/10.1021/ic500986x | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

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were previously classiﬁed as “unique” Abs spectra based

primarily on the unusual appearance of the γ region. As in

revealed that the spectral changes in the α/β region that occur

4

in response to a DMB → H O lower ligand substitution reﬂect

2

those latter spectra, the pair of features at ∼19 000 and 20 000

a stabilization of the HOMO relative to the LUMO, the extent

−1

cm in the Abs spectrum of NOCbl also appear to correspond

to the origin and ﬁrst member of a vibrational progression in a

totally symmetric breathing mode of the corrin ring. In support

of these assignments, the corresponding features are of the

same sign in both the CD and the MCD spectra of NOCbl

of which depends on the σ-donating strength of the upper axial

20

ligand. As the blue shift of the α/β bands from NOCbl to

+

−1

NOCbi (∼1000 cm ) is considerably smaller than the shift

+

−1

4

observed from MeCbl to MeCbi (∼2500 cm ), NO may

appear to be a weaker σ-donating ligand than a methyl group.

However, since the α/β bands of NOCbl occur at highe₄r

energies than those of MeCbl and all other alkylcobalamins,

(

Figure 2, middle and bottom). The other bands in the visible

region of the NOCbl Abs spectrum are due to at least one or

more additional electronic transitions, as indicated by their

opposite signs in the CD spectrum. Consistent with its

classiﬁcation as a unique Abs spectrum, several bands that

arise from distinct electronic transitions can be identiﬁed in the

γ region, including two similarly intense features centered at 31

+

their small blue shifts from NOCbl to NOCbi could also be

due to the fact that the DMB moiety is only weakly interacting

with the Co ion in the former species, as suggested by the

unusually long Co−N(DMB) bond observed in the crystal

16

structure of NOCbl.

00 cm⁻with a low-energy shoulder at 28 000 cm .

1

−1

Intriguingly, while the Abs and CD spectra of NOCbi⁺

obtained at 4.5 and 300 K diﬀer insigniﬁcantly with respect

to band positions (Figure 3, top and middle), the Abs spectrum

of NOCbl collected in ﬂuid solution at 300 K shows the α/β

0

Upon substitution of the DMB ligand of NOCbl with a more

+

−1

weakly σ-donating water molecule in NOCbi , a ∼1000 cm

blue shift of the α/β bands is observed (Figures 3, top).

−1

bands blue-shifted by ∼850 cm from their positions at 4.5 K

(

Figure 2, top). Signiﬁcant temperature-dependent changes are

also observed in the CD spectrum of NOCbl, most notably a

drastic decrease in the intensity of the lowest-energy, negatively

−1

signed feature at ∼18 000 cm and the appearance of a weak,

positively signed feature at even lower energy (Figure 2,

middle). As a result, the room-temperature Abs and CD spectra

+

of NOCbl quite closely resemble those of NOCbi .

Collectively, these results suggest that by increasing the

temperature from 4.5 to 300 K, the base-on to base-oﬀ

equilibrium for NOCbl changes, favoring dissociation of the

N(DMB) ligand at high temperatures. However, consistent

2

with published NMR results, a sizable fraction of NOCbl must

remain in the base-on state even under ambient conditions,

+

since the 300 K Abs spectra of NOCbl and NOCbi are not

superimposable (see Supporting Information, Figure S1). Yet,

while the NMR data were interpreted to indicate that at room

temperature ∼67% of the NOCbl molecules are in the base-on

state, the traces obtained by adding the 4.5 K Abs or CD

+

spectra of NOCbl (scaled by 0.67) and NOCbi (scaled by

0

.33) diﬀer from the 300 K Abs and CD spectra of NOCbl.

This ﬁnding suggests that the Co−N(DMB) bond of base-on

NOCbl is longer at 300 K than it is at 4.5 K.

II. Resonance Raman Data. The 77 K rR spectrum of

NOCbl obtained with laser excitation at 488.0 nm (20,490

−1

cm ) shows strong enhancement of four features between

−

1

450 and 1650 cm . On the basis of our recent study of the

44

vibrational properties of vitamin B and its reduced forms, all

12

of these features can be attributed to corrin-based vibrational

modes. Three of them (termed ν −ν ) arise from totally

S1

S3

Figure 3. Abs (top), CD (center), and 7 T MCD (bottom) spectra at

various temperatures of NOCbi , a spectroscopic model of base-oﬀ

NOCbl.

+

symmetric modes (assuming an eﬀective C symmetry, with the

s

^p^s^e^u^d^o^mⁱ^r^r^o^r^p^l^aⁿ^e^b^eⁱⁿ^g^o^rⁱ^eⁿ^t^e^d^a^l^oⁿ^g^t^h^e^C^o^aⁿ^d^C10

atoms and perpendicular to the corrin ring plane) and are thus

particularly strongly resonance enhanced, while the fourth (ν_a_s)

is associated with an asymmetric stretching mode. A more

detailed analysis of these modes within the framework of our

DFT frequency calculations is provided below.

Additionally, a positively signed feature at 18 500 cm⁻appears

1

+

in the CD spectrum of NOCbi that has no counterpart in the

NOCbl spectrum, while the prominent negative feature at 19

0

00 cm⁻decreases in intensity and shifts to higher energy

1

A comparison of the low-energy regions of the rR spectra of

15

(

Figure 3, middle). Because the MCD spectra of NOCbl and

NOCbi are essentially temperature-independent in the 4.5 to

0 K range (Supporting Information, Figures S4 and S5), it can

be concluded that both species possess diamagnetic (S = 0)

ground states, consistent with a Co(III)/NO oxidation state

NOCbl and its NOCbl isotopomer reveals an isotope-

+

−1

sensitive feature at 532 cm (Figure 4, top), a region where the

5

Co−NO stretching and Co−N−O bending modes are

10

15

expected to occur. Subtraction of the NOCbl from the

−

NOCbl trace yields a diﬀerence spectrum that shows an

1

−1

14

assignment. Previous studies of other Co(III) corrinoids have

apparent shift of the 532 cm⁻feature to 496 cm upon NO

D

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Inorganic Chemistry

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Figure 4. Low-energy region of rR spectra of NOCbl and its ¹⁵NO-

enriched isotopomer in the base-on (top) and base-oﬀ (bottom)

−1

conformations, obtained at 77 K with 488 nm (20 491 cm ) laser

excitation. A diﬀerence spectrum for each conformation is included

below the two data sets to highlight the isotope-sensitive vibrational

features. Ice peaks are marked with asterisks.

Figure 5. High-energy region of rR spectra of NOCbl and its ¹⁵NO-

enriched isotopomer in the base-on and base-oﬀ conformations,

obtained at 77 K with 488 nm (20 491 cm ) laser excitation. A

diﬀerence spectrum for each conformation is included below the two

data sets to highlight the isotope-sensitive vibrational features.

−1

→ ¹⁵

NO substitution. An analogous isotope-sensitive feature is

present in the rR spectrum of base-oﬀ NOCbl (our model of

+

NOCbi , Figure 4, bottom traces), though in this case a much

better-resolved diﬀerence spectrum is obtained. Closer

examination of this diﬀerence spectrum clearly discloses the

presence of a shoulder on the low-energy side of the positive

feature, suggesting that two isotope-sensitive modes actually

occur in this region. Indeed, a Gaussian deconvolution of the rR

spectra in Figure 4 reveals that the vibrational modes of base-on

III. Computational Studies. DFT has been used by us with

great success for the study of corrinoids in their Co(III),

19

Co(II), and Co(I) oxidation states. While diﬀerent func-

tionals have been shown to provide variable descriptions of

45

spectroscopic properties, careful evaluations of the computa-

tional results on the basis of experimental data have aﬀorded a

detailed understanding of the chemical and spectroscopic

properties of a large number of diﬀerent corrinoid spe-

−1

and base-oﬀ NOCbl at 515 and 532 cm shift to 500 and 521

−

1

14

15

cm , respectively, upon NO → NO substitution (see

Supporting Information, Figure S9). In a previous rR study of

NOCbl, a single isotope-sensitive peak was observed at 514

1

9,4,23,21,27,46

cies.

From a recent study of MeCbl, Kozlowski

and co-workers have suggested that pure GGA functionals,

rather than hybrid functionals, provide DFT results more

−1

14

15

cm that shifted to 496 cm upon NO → NO substitution.

However, the spectral resolution of these published data

appears to be relatively low, as only two high-energy (>1,500

4

7

consistent with correlated wave function-based methods.

20

48

Furthermore, work by our group and others has revealed

that complete cofactor models should be used to obtain

accurate geometric structures of cobalamins, while truncated

models can be used to compute various spectroscopic

parameters. In light of these ﬁndings, we performed geometry

−1

cm ) features associated with corrin-based modes could be

1

0

identiﬁed, compared to the four features that are readily

apparent in our spectra (Figure 5). Additionally, considering

that mode coupling typically leads to lower isotope shifts than

−1

+

expected, the reported isotope shift of 18 cm , which largely

optimizations of complete NOCbl and NOCbi species using

−

1

exceeds the 14 cm decrease in vibrational frequency

calculated using a harmonic oscillator model for a localized

Co−N stretching mode, seems unreasonably large.

the pure PBE functional. The resulting structures were then

suitably truncated to predict the Abs and CD spectra of these

species using TDDFT, as well as to analyze their vibrational

properties (see Methods for details). For comparison, we also

carried out full geometry optimizations of these truncated

models and calculated their Abs and CD spectra.

i. Geometries. The most signiﬁcant diﬀerences between the

X-ray crystal structure and our DFT-optimized model of

NOCbl include the Co−N(DMB) bond distance and the

folding of the corrin ring. While the crystal structure of NOCbl

shows an unusually long Co−N(DMB) bond of 2.35 Å, our

computation predicts this bond to be elongated by an

The high similarity between the low-energy regions of the rR

spectra of base-on and base-oﬀ NOCbl (Figure 4) indicates that

the Co−NO bonding interaction is largely unperturbed by the

DMB → H O lower-ligand substitution. Consistent with this

2

conclusion, the rR spectra of ﬂuid solution samples of base-on

and base-oﬀ NOCbl are very similar to each other as well as to

the corresponding 77 K spectra (see Supporting Information,

Figures S7 and S8), despite the thermochromism exhibited by

NOCbl.

E

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+

Table 1. Relevant Structural Parameters of NOCbl and NOCbi , as Derived Experimentally or Obtained Computationally

0

θ(LA) (⁰)

0

species

NOCbl

model

Co−X_u_p_p_e_r(Å)

Co−X_l_o_w_e_r(Å)

N−O (Å)

∠Co−N−O ( )

Φ(SA) ( )

a

crystal

1.91

1.87

1.85

2.35

2.51

2.61

3.58

2.98

1.14

1.19

1.18

119.4

119.8

119.3

120.9

120.2

12.4

9.2

3.3

9.2

3.6

7.5

8.9

full

truncated

full

5.8

NOCbi⁺

10.43

4.5

truncated

a

From ref 16.

additional 0.16 Å, to 2.51 Å (see Table 1). A further elongation

of this bond by 0.1 Å, along with a tilting of the DMB ring

plane relative to the Co−N(DMB) bond vector, occurred

during the geometry optimization of our truncated model

lacking the propionamide side chains. Although DFT geometry

optimizations of cobalamin models consistently overestimate

Owing to the overall good agreement between the X-ray

crystal structure and DFT-optimized model of NOCbl, a closer

examination of the energy-minimized NOCbi⁺model is

warranted. As expected, replacing the bulky DMB base with a

water molecule in the lower axial position causes sizable

changes to both of the corrin fold angles (Table 1). More

2

0,49

importantly, the optimized Co−O(H ) distance in our

the Co−N(DMB) bond distance by ∼0.1 Å,

the elongated

2

+

complete NOCbi model is 3.58 Å, suggesting that the Co

Co−N(DMB) bonds in our models may also stem from the

neglect of intermolecular interactions that modulate the length

of this bond in the solid state and in solution. Regardless, these

ﬁndings support the notion that the Co−N(DMB) bond of

NOCbl is very weak and can thus be stretched, or potentially

even broken, quite readily.

ion resides in an eﬀective ﬁve-coordinate ligand environment.

Although this distance shortens by 0.60 Å upon removal of the

propionamide side chains, the “lone pairs” on oxygen in our

truncated model are not actually oriented properly to engage in

a bonding interaction with the Co ion. A closer inspection of

the complete NOCbi⁺model reveals that several of the

Distortions of the corrin ring can be assessed on the basis of

the long-axis and short-axis fold angles, θ(LA) and Φ(SA),

respectively. θ(LA) is deﬁned here as the angle between the

plane containing the N , C , C , C , and N atoms and the

propionamide side chains form a cage around the H O

2

molecule. The fact that removing these side chains causes a

large rearrangement of the water molecule suggests a role of the

propionamide groups in modulating the ligation of the lower

ligand by imposing steric constraints and/or providing a

solvent-protected pocket for the ligand.

A

4

5

6

B

plane containing the N , C , C , C , and N atoms, while

C

14

15

16

D

Φ(SA) corresponds to the angle between the planes containing

the N , C , C , and N atoms and the N , C , C , C , and N

D

19

1

A

B

9

10

11

C

Finally, as expected in light of the strong Co−NO bonding

atoms (see Figure 1 for the atom numbering scheme used). As

such, θ(LA) correlates with the amount of “butterﬂy fold” of

the corrin ring and is thus particularly sensitive to the

positioning or removal of the bulky DMB ligand. Alternatively,

Φ(SA) reﬂects the extent of “ring ruﬄing” due to steric

constraints imposed on the propionamide and methyl groups

on the A and D rings. As shown in Table 1, the θ(LA) values

derived from the crystal structure and DFT-optimized complete

model of NOCbl vary quite substantially, as expected from the

sizable variation in the corresponding Co−N(DMB) bond

distances. In contrast, the Φ(SA) values are nearly identical,

suggesting that the corrin ring conformation observed

experimentally is minimally aﬀected by crystal-packing eﬀects

and/or intermolecular H-bonding interactions. The fact that

DFT predicts both fold angles to decrease quite substantially

upon removal of the propionamide side chains is consistent

with our previous ﬁnding that distortions along the θ(LA) and

interaction in NOCbl, the Co−NO unit is largely unaﬀected by

the DMB → H O lower-ligand substitution (Table 2, see NBO

2

Table 2. Summary of the Results Obtained from a Natural

+

Population Analysis (NPA) for the NOCbl, NOCbi , and

O Cbl Truncated Models

2

natural charge

X_u_p_p_e_r

natural bond order

species

Co

X_l_o_w_e_r

Co−X_u_p_p_e_rCo−X_l_o_w_e_rX_u_p_p_e_r−O

NOCbl

0.82

0.84

0.15

0.16

−0.47

−0.96

−0.42

0.82

0.84

0.47

0.07

0.02

0.25

1.98

0.75

_N_O_C_b_i+

O Cbl

2

−0.14

analysis for more details), with the most notable change being a

small (0.02 Å) decrease in the Co−N(O) bond distance. A

similar shortening was predicted previously for the Co−

+

4

C(methyl) bond upon conversion of MeCbl to MeCbi .

50

Φ(SA) coordinates require very little energy.

Hence, despite the long Co−N(DMB) bonds in NOCbl and

MeCbl, the base does play a small role in modulating the upper

axial bonding interactions in these species.

Despite these modest discrepancies between the experimen-

tally determined and DFT-optimized NOCbl structures, the

metric parameters for the Co−NO unit agree very well.

Speciﬁcally, the Co−N(O) distances and Co−N−O bond

angles are identical to within 0.04 Å and <1°, respectively.

Notably, the Co−N(O) bond is signiﬁcantly shorter than the

upper axial ligand−Co bond distances reported for all other

ii. TDDFT Results. The computed Abs spectrum for the

NOCbl model derived from the complete structure correctly

predicts the major features observed experimentally, including

the presence of two clusters of intense features near 20 000

−1

cm and 31 000 cm , corresponding to the α/β region and

the γ region, respectively (Figure 6, top). The computed CD

spectrum is also in reasonable agreement with our experimental

spectrum, both exhibiting a derivative-shaped feature associated

with two oppositely signed transitions centered at 20 000 cm⁻

and a series of features with alternating signs in the γ region

(Supporting Information, Figure S10, top). While the

computed Abs and CD spectra for the optimized truncated

5

1,52

Co(III)Cbl species,

suggesting that this bond is exception-

ally strong. Although the computation appears to overestimate

the N−O bond distance by 0.05 Å, note that the value of 1.14 Å

determined experimentally is surprisingly small, given that (i)

1

53

the N−O bond distance in nitric oxide is 1.15 Å and (ii) the

NO ligand in NOCbl acquires a partial negative charge and

thus increased π-antibonding electron density (vide infra).

F

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+

Figure 6. Abs spectra of NOCbl (top) and NOCbi (bottom) collected at 4.5 K (dotted lines) superimposed on the TD-DFT results. The TD-DFT

−1

computed transitions (vertical sticks) were convoluted with Gaussian bands with a constant fwhm of 1250 cm to obtain the predicted spectra

+

plotted in dark blue for NOCbl and in light blue for NOCbi . In each case, the EDDM for the α-band transition is shown on the left, where regions

of loss and gain of electron density are shown in gray and gold, respectively. The calculated spectra were uniformly red-shifted by 2200 cm⁻to

1

facilitate a direct comparison with the experimental results.

NOCbl model are similar to those obtained with the modiﬁed

in terms of atomic displacement vectors is provided in Figure 7.

−1

complete model, the α/β bands are blue-shifted by 900 cm ,

and the lowest-energy, negatively signed feature in the CD

spectrum is considerably weaker, which agrees less well with

our experimental data (Supporting Information, Figure S11).

The three modes that retain the approximate C symmetry of

s

+

NOCbi are expected to be most strongly resonance-enhanced,

because only totally symmetric modes can couple to electronic

transitions and thus gain rR intensity via the Franck−Condon

5

5,56

Upon DMB → H O lower-ligand substitution, the α/β bands

mechanism.

Even though DFT methods typically over-

2

in the TDDFT-computed Abs and CD spectra blue shift by

estimate vibrational frequencies, the agreement between the

experimental and computed frequencies for these modes is

excellent, thus permitting a straightforward assignment of the

−1

∼

3500 cm , and the negative CD feature at 20 000 cm

decreases in intensity, causing an apparent blue shift of the

−1

derivative-shaped feature to 21 000 cm (Figure 6, bottom,

Supporting Information, Figure S10, bottom). As expected on

relevant vibrational features. Speciﬁcally, the 1497 cm feature

observed experimentally is assigned to the formally totally

−1

the basis of the negligible Co−O(H ) bonding interactions in

symmetric ν_s₁mode predicted at 1506 cm , which involves

nuclear motion primarily along the short axis of the corrin ring.

Alternatively, the 1541 and 1603 cm⁻features in the

2

+

the two diﬀerent NOCbi models, the Abs and CD spectra

1

predicted for these species are almost identical (Supporting

Information, Figure S12). Overall, the computed Abs spectra

experimental rR spectra are attributed to the ν and ν_s₃

s2

+

−1

for both NOCbi models agree quite well with the experimental

modes predicted at 1554 and 1613 cm , respectively. These

spectrum across the entire spectral range investigated, except

modes mainly entail C−N stretching motion along the long axis

of the corrin ring and symmetric methine stretching motion,

respectively. The remaining feature observed at 1572 cm⁻is

−1

for the presence of a small feature at ∼15 000 cm that is not

observed experimentally. In contrast, the computed CD spectra

only modestly reproduce the experimental spectrum, which is

not unexpected because magnetic-dipole allowed transitions

1

then assigned to the antisymmetric ν mode predicted at 1585

as

−1

cm . While the large depolarization ratio (0.71) computed for

ν_a_ssuggests that this mode should not be resonance-enhanced,

it is predicted to carry signiﬁcant oﬀ-resonance Raman intensity

and to entail large displacements of corrin ring atoms, thus

facilitating intensity enhancement via Herzberg−Teller cou-

54

pose a signiﬁcant challenge for TDDFT computations.

iii. Vibrational Frequencies. While considerable diﬀerences

are observed between the experimental Abs, CD, and MCD

+

spectra of NOCbl and NOCbi (cf. Figures 2 and 3), the rR

5

5,56

spectra obtained for these species show minimal diﬀerences

with respect to the frequencies of the major corrin and Co−

NO-based modes (vide supra). For this reason, and for

computational practicality, the truncated NOCbl and NOCbi⁺

models were employed in our DFT-assisted vibrational analysis.

The DFT-computed oﬀ-resonance Raman spectra for these

pling.

Overall, these assignments are consistent with our

vibrational analysis recently carried out for CNCbl and its

44

reduced derivatives.

Our calculations for NOCbi also predict two modes at 569

+

and 585 cm⁻with NO → NO isotope shifts of Δν

1

14

15

=

N15

−1

−7.3 and −6.0 cm , respectively. These predictions agree

reasonably well with the frequencies (515 and 532 cm ) and

isotope shifts (Δν

experimentally. Both of these modes involve large atomic

−1

models exhibit four relatively intense features in the 1450−

650 cm⁻region (Supporting Information, Figure S13 and

1

= −15 and −11 cm ) observed

−1

1

N15

S14). A graphical representation of the corresponding normal

+

modes of NOCbi (which are very similar to those of NOCbl)

displacements along the Co−N(O) stretching and Co−N−O

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Figure 8. Isosurface plots of the relevant MOs of NOCbl. The dashed

line separates occupied from empty orbitals.

Figure 7. Eigenvector representations of the relevant normal modes

energy, whereas the Co 3d -based MO (distributed over MOs

xy

+

for the truncated NOCbi model, as obtained with DFT. The

#

167 and #168 due to mixing with another, energetically

1

4

15

computed frequency (ν), isotope shift upon NO → NO

substitution (Δν_N₁₅), depolarization ratio (ρ), oﬀ-resonance Raman

intensity (I), and assignment are shown for each mode.

proximate MO) is strongly σ-antibonding with respect to the

Co−N(corrin) bonds and therefore highest in energy (note

that the x and y axes are rotated by 45° about the z axis from

their usual orientations due to the Co−N(corrin) π-bonding

interactions). Intriguingly, the HOMO and LUMO of NOCbl

derive mostly from Co 3d and NO π*orbitals, in contrast to the

case of alkylcobalamins (e.g., MeCbl) where these MOs are

bending coordinates and are coupled out of phase and in phase,

respectively, with corrin ring breathing modes. As such, the

distinction between bending and stretching modes becomes

1

4

15

4

ambiguous in this case. Only one additional NO → NO

primarily corrin π/π*-based. Speciﬁcally, the HOMO of

NOCbl contains 28% Co 3d_zand 40% NO π * orbital

−

1

2

isotope-sensitive mode is computed at 1688 cm , correspond-

ing to a relatively pure N−O stretch. While this mode is

predicted to be strongly IR active, it carries negligible intensity

in the computed oﬀ-resonance Raman spectrum (Supporting

Information, Figures S13 and S14, respectively). This

observation, as well as the lack of mechanical coupling between

the N−O and corrin ring stretches and the weak electronic

coupling between the NO moiety and corrin π system (see

below) predicted computationally are consistent with the

absence of a feature attributable to the N−O stretch in our

∥

contributions (to diﬀerentiate between the two NO π* orbitals,

the one with its lobes roughly parallel to the Co−N(O) bond

axis will be denoted as π * and the other as π *), making it

∥

⊥

strongly σ-bonding with respect to the Co−N(O) bond (see

Figure 8 for MO plots and Supporting Information, Table S1

for compositions). Alternatively, the LUMO corresponds to the

π-antibonding combination of the Co 3d_y_zand the NO π_⊥*

orbitals (15% and 78%, respectively), with the bonding

counterpart being considerably lower in energy (MO #162).

Upon replacement of the axial DMB ligand of NOCbl with a

+

experimental rR spectra of NOCbl and NOCbi .

+

iv. DFT-Computed Molecular Orbital Diagrams. The good

agreement between the experimental and computed Abs, CD,

and vibrational data presented above indicates that DFT

successfully models the salient bonding interactions present in

more weakly σ-donating water molecule in NOCbi , MOs with

2

z

large Co 3d orbital contributions (MOs #163, #164, #167,

and #168 of NOCbl, corresponding to MOs #128, #130, #133,

+

and #134 of NOCbi , see Figure 9) are stabilized by ∼0.1 eV

+

2

NOCbl and NOCbi . For both species, the relative energies of

relative to the Co 3d -based MO, which was chosen as the

x −y

the Co 3d-based MOs reﬂect the strongly σ- and weakly π-

reference point because its composition remains essentially

donating nature of the tetradentate corrin ligand. Among these

unchanged from NOCbl to NOCbi⁺(see Supporting

2

z

orbitals (see Figure 8), the Co 3d -based MO (#158 in the

Information, Tables S1 and S2). In particular, the Co 3d /

x −y

+

case of NOCbl) is essentially nonbonding and thus lowest in

NO π *-based HOMO of NOCbi (#130) loses the σ-

∥

H

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responsible for the α/β bands. The largest changes in electron

density occur within the C −C −C fragment of the corrin

9

10

11

ring, consistent with the strong rR enhancement of the ν

s1

mode observed experimentally (Figure 5).

The EDDMs for the dominant transitions in regions ii

excited states 21, 23, and 26) and iii (excited state 37) display

(

large changes in electron density around the DMB and NO

ligands, due to one-electron excitations from MOs with

signiﬁcant orbital contributions from the DMB group. Lastly,

regions iv and v are dominated by several intense corrin π → π*

transitions that also cause a moderate electron density

redistribution at the Co center as a result of the sizable Co

3

d orbital character in the donor and acceptor MOs. The

prediction of numerous intense features in the γ region of the

computed Abs spectrum, as opposed to a single, relatively sharp

4

band as is usually observed for Co(III)Cbl species, is

consistent with the unique Abs spectrum obtained exper-

imentally for NOCbl.

+

From NOCbl to NOCbi , TD-DFT predicts the most

intense feature in the visible region of the Abs spectrum to blue

−1

shift by 3500 cm , in qualitative agreement with the ∼1000

−1

cm shift of the α/β bands observed experimentally (Figure

). However, additional features are present in the near-IR

6

region of the computed Abs spectrum that have no discernible

counterparts in the experimental spectrum. As revealed by their

EDDMs (Supporting Information, Figure S15), the transitions

associated with the ﬁrst two features in regions 0 and ia cause a

large electron redistribution within the Co−NO moiety (see

Table 4). The fact that analogous features are not observed

experimentally indicates that TDDFT incorrectly predicts the

intensities and/or energies of these charge transfer (CT)-type

+

Figure 9. Isosurface plots of the relevant MOs of NOCbi . The dashed

line separates occupied from empty orbitals.

+

transitions of NOCbi ; similar cases where TDDFT fails to

properly describe CT excited states are well-documented in the

5

7,58

antibonding interaction with the lower axial ligand and gains

literature.

The remaining changes in the computed Abs

+

contributions from the corrin π MO. This mixing causes a

spectra from NOCbl to NOCbi pertain to a signiﬁcant

intensity redistribution among the dominant features in the γ

region, which is again in good qualitative agreement with our

experimental results.

7

large stabilization of the corrin π -based MO #128 relative to

7

+

the corrin π -based MO #132 of NOCbi (corresponding to

8

MOs #163 and #166 of NOCbl).

v. TDDFT-Assisted Spectral Assignments. Given the

satisfactory agreement between the experimental and computed

spectroscopic data of NOCbl and NOCbi , it is reasonable to

Because our TDDFT computations reproduce the key

diﬀerences between the Abs spectra of NOCbl and NOCbi⁺

observed experimentally quite well, the computed MO

diagrams can be used as the basis for exploring the electronic

structural origin of these diﬀerences. As described above, the

+

assign the key Abs features of these species within the

framework of the DFT-based MO descriptions as provided by

the TDDFT results. Because in the TDDFT formalism

electronic transitions are described as linear combinations of

one-electron excitations between occupied and virtual MOs, it

becomes diﬃcult to identify the nature of a given transition in

cases where multiple excitations make signiﬁcant contributions.

One approach to overcome this limitation involves computing

electron diﬀerence density maps (EDDMs), which provide a

visual representation of the changes in electron density

accompanying an electronic transition. On the basis of such

EDDMs (Figure 6), the electronic transitions producing the

dominant contributions to the TDDFT-computed Abs spectra

can be assigned as shown in Table 3 for NOCbl and Table 4 for

DMB → H

with signiﬁcant Co 3d

other MOs. Of particular importance with respect to the α/β

band transition is the stabilization of the corrin π -based donor

MO relative to the corrin π -based acceptor MO from NOCbl

O ligand substitution causes a stabilization of MOs

2

orbital contributions relative to the

z

7

8

+

(MOs #163 and #166) to NOCbi (MOs #128 and #132),

which readily explains the blue shift of the α/β bands observed

experimentally (Figures 2, 3, and 6). Interestingly, in the case of

alkylcobalamins that also possess a strong σ-donor ligand in the

upper axial position, the blue shift of the α/β bands in response

to DMB → H O ligand substitution is considerably larger than

2

−1

it is in the case of NOCbl; for example, ∼2500 cm for

+

4

−1

NOCbi . In the case of NOCbl, the ﬁve lowest-energy

MeCbl versus ∼1000 cm for NOCbl. This diﬀerence can be

understood in terms of the larger σ-donation from the upper

ligand in NOCbl than in alkylcobalamins, which leads to a

weaker Co−N(DMB) bond and, thus, to less pronounced

changes in the electronic structure upon lower-ligand

substitution in the former species. In fact, our computed MO

description for NOCbl bears some intriguing similarities to that

developed previously for Co(II)Cbl, especially with regard to

transitions carrying signiﬁcant Abs intensity give rise to one

broad feature in region i of the computed Abs spectrum (Figure

6

1

, top), with two transitions (involving excited states 12 and

5) having the largest oscillator strengths. On the basis of its

EDDM, transition 12 primarily entails a one-electron excitation

from the corrin π -based MO #163 to the corrin π -based MO

7

8

#

166, characteristic of the transition that is generally

I

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−1

Table 3. TDDFT-Calculated Energies (in cm ), Polarizations, Oscillator Strengths, and Percent Contributions from One-

Electron Excitations for the Major Electronic Transitions of NOCbl (Band Assignments Are Shown in Figure 6). Donor and

Acceptor MOs Are Labeled According to Their Predominant Co/Corrin-π Character, with Percent Co Contributions Shown in

Parentheses

the energies of the Co 3d-based MOs relative to the corrin π/

π*-based MOs. This observation indicates that the high

To overcome this challenge, we resorted to the NBO formalism

within which the calculated electron density is partitioned into

chemically intuitive bonding MOs and electron pairs so as to

generate a bonding description closely adhering to the Lewis

2

3

degree of σ-donation from the occupied NO π * orbital into

∥

2

z

the formally unoccupied Co 3d orbital leads to a signiﬁcant

5

9

decrease in the eﬀective nuclear charge experienced by the Co

structure formalism. Because the Co−N(O) bonding

+

3d orbitals in NOCbl. However, the NO → Co charge

interactions in NOCbl and NOCbi are essentially identical

donation is partially compensated for by the strong π-

as judged on the basis of our experimental rR data and DFT

backbonding interaction involving the doubly occupied Co

results (vide supra), the following analysis will focus on the

electronic structure of NOCbi . The relevant NBOs, labeled by

+

3

d_y_zorbital and formally empty NO π * orbital. A more

⊥

detailed analysis of the salient Co−NO bonding interactions

their primary orbital contributors and classiﬁed either as

bonding (BD) or antibonding (BD*) with respect to the Co−

NO bond, are shown in Figure 10 (left). As expected on the

within the NBO formalism is presented next.

vi. NBO-Based Co−NO Bonding Description. While a

relatively straightforward correlation can be established

between the DFT/TDDFT-predicted and experimentally

2

basis of the DFT results described above, the Co 3d BD-NBO

z

is characterized by a highly covalent Co−NO σ-bonding

+

observed properties of NOCbl and NOCbi , a direct evaluation

interaction. However, shortcomings of the Lewis structure-like

+

of the key Co−NO bonding interactions is complicated by the

partial delocalization of the relevant MOs over the corrin ring.

description for NOCbi are evident from the relatively large

occupancies of several formally empty orbitals, most

J

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−1

Table 4. TDDFT-Calculated Energies (in cm ), Polarization, Oscillator Strengths, and Percent Contributions from One-

+

Electron Excitations for the Major Electronic Transitions of NOCbi (Band Assignments Are Shown in Figure 6). Donor and

Acceptor MOs Are Labeled According to Their Predominant Co/Corrin-π Character, with Percent Co Contributions Shown in

Parentheses

+

importantly the antibonding Co 3d_x_yBD*-NBO and Co 4s

BD*-NBO, as well as the occupancies of considerably less than

The relevant NLMOs for NOCbi are shown in Figure 10

(right), and their compositions are provided in Table 5.

Inspection of the Co 3d BD-NLMO discloses a very covalent

σ-bonding interaction between the Co ion and NO (58% Co

d and 42% N(O) 2p orbital contributions, see Table 5), as

anticipated on the basis of the canonical MO and NBO

compositions. Intriguingly, while the Co 3d BD-NLMO as

2

z

2

of some of the formally doubly occupied orbitals. These

observations indicate that deviations from a single Lewis

2

3

z

structure, treated within the NBO framework as donor−

60

acceptor hyperconjugation interactions, are important for

2

z

+

properly describing the bonding interactions in NOCbi . These

2

well as the nonbonding Co 3d

and 3d_x_zNLMOs are very

x −y

interactions can be fully accounted for by a transformation of

the canonical MOs to a set of natural localized MOs (NLMOs),

which are constructed to retain a large amount of NBO

character. These NLMOs possess integer occupancies, like the

canonical MOs, and thus provide a useful connection between

similar to their corresponding NBOs, the 3d_y_zBD-NLMO

shows distinct diﬀerences. The composition of this NLMO

reveals that it only retains 83.1% of “pure” Co 3d_y_zNBO

character (in comparison to >95% for the other Co-based

NLMOs) due to a sizable π-backbonding interaction between

the Co 3d orbital and the NO π * orbital (Figure 10, center

5

9,61,62

the canonical MOs and the NBOs.

yz

⊥

K

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+

Figure 10. Isosurface plots of the metal-based NBOs (in blue/gray) and corresponding NLMOs (in orange/gray) of NOCbi . Occupancies are

shown below each orbital. The composition of the NBOs and their percent contributions to the NLMOs are provided in Table 4

Table 5. Summary of NBO and NLMO Results for NOCbi⁺.

Energies, Occupancies, and Percent Co/N_N_OCompositions

of the Co-Based NBOs Are Shown, in Addition to the

Percent Contributions of These NBOs to the Corresponding

NLMOs. Low-Occupancy NBOs, which Correspond to

thus an eﬀective oxidation state of the Co ion. As the number of

d electrons is sensitive to the covalency of the metal−ligand

bonding interactions as well as the delocalization of the 3d

orbitals, the following approach was used to estimate this value

(see Supporting Information for additional details). First, the %

NBO composition of each NLMO (see Table 5) was used to

calculate the fractional occupancy of the Co 3d-based NBO(s)

contributing to this particular NLMO. This electron count was

then partitioned into Co 3d-based and ligand-based contribu-

tions, estimated based on the %Co 3d contributions to each

NBO. Finally, the fractional Co 3d electron counts for all

NLMOs obtained using this procedure were added up to

estimate the total number of Co 3d electrons. Using this

approach, an eﬀective number of Co 3d electrons of 6.27 was

a

Formally Empty Orbitals, Are Highlighted in Red

+

obtained for NOCbi , larger than a value of 6 expected for a

Co(III) species. This result corroborates the conclusion drawn

from the energies and compositions of the canonical MOs

regarding the relatively high Co(II) character in the electronic

a

NA indicates values not availalable from NLMO composition

analysis, but estimated to be close to 100%.

+

structures of NOCbl and NOCbi . Thus, while the large

columns). Since the Co 3d orbital only mixes with the NO

^π⊥^*^o^r^bⁱ^t^a^l⁽^Fⁱ^g^u^r^e¹⁰⁾^,^t^h^e^a^m^o^uⁿ^t^o^f^c^h^a^r^g^e^b^a^c^k^d^oⁿ^a^tⁱ^oⁿ

2

yz

amount of electron donation from NO to Co in the Co 3d_z

BD-NLMO is partially oﬀset by the strong π-backbonding

from Co to NO can be estimated to be ∼0.32 electrons.

interaction in the Co 3d_y_zBD-NLMO, our NBO/NLMO

Because of the presence of this π-backbonding interaction, the

Co ion does not retain a Co(II)-like electronic structure as

might be anticipated on the basis of the large σ donation from

−

analysis clearly shows that a Co(III)−NO description for

+

NOCbi , and by analogy NOCbl, is inaccurate. Rather, these

species are better described as resonance hybrids with both

2

z

−

•

the NO π * orbital to the Co 3d orbital. Lastly, the

∥

Co(III)−NO and Co(II)−NO limiting structures being

2

^uⁿ^o^c^c^u^pⁱ^e^d^C^o³^dz BD*-NLMO and Co 3d_x_yBD*-NLMO

are σ-antibonding with respect to the corrin ring and the axial

ligand orbitals, as expected, and retain >95% of NBO character

major contributors.

DISCUSSION

(

see Table 5 and Figure 10, outside columns).

■

The NBO/NLMO results can also be used to estimate the

Given the roles of NO as a blood-pressure regulator,

neurotransmitter, and second messenger, as well as a

6

3

number of electrons in the Co 3d atomic orbitals (AOs) and

L

dx.doi.org/10.1021/ic500986x | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Figure 11. Isosurface plots of the metal-based NLMOs of O Cbl. The α orbitals (majority spin, on left) are shown in orange/gray, while β orbitals

2

(

minority spin, on right) are shown in yellow/gray. The horizontal dashed line separates singly occupied from empty orbitals. NLMOs are labeled

according to the Co-based NBO providing the largest contribution. The compositions of the NLMOs in terms of the corresponding NBOs are

provided in Table 5

6

3

cytotoxic agent in immunological response, the ability of

the Co−NO bonding interaction, as evidenced by our rR data,

TDDFT results, and NBO analysis. The origin and implications

of these ﬁndings are discussed below.

Co(II)Cbl to scavenge this molecule in vivo to form NOCbl is

9

,10

of considerable interest.

Additionally, NOCbl has been

shown to be potentially useful for targeted NO delivery as a

Co−NO Bonding. Collectively, our spectroscopic and

6

4,12

chemotherapeutic or vasodilating agent,

since its Co−NO

computational data reveal that NOCbl is inadequately

−

bond can be broken under physiological conditions for

controlled NO release. Thus, elucidating the nature of the

bonding interaction between the NO ligand and the Co center

in NOCbl represents an important step toward the develop-

ment of an improved understanding of the chemical and

biological properties of this molecule. Previous reports have

highlighted the unique structural properties of NOCbl relative

to other biologically relevant cobalamins, most notably the

presence of an unprecedentedly long Co−N(DMB) bond in

this species. However, because NOCbl exhibits similar

spectroscopic signatures as the well-characterized alkyl-Co(III)-

described as a Co(III)−NO species, because signiﬁcant charge

donation from the NO ligand notably alters the eﬀective

nuclear charge of the metal center. Compared to the methyl

group of MeCbl, the NO ligand of NOCbl is an even stronger

2

z

σ-donor, therefore inducing additional Co 3d orbital character

into the HOMO and further enhancing the Co−N(DMB) σ-

antibonding interaction in this orbital. Our DFT-computed

2

MO description indicates that the formally unoccupied Co 3d_z

orbital contributes as much as 29% to the HOMO of NOCbl,

4

as compared to 7% in MeCbl. This prediction is consistent

with the observed lengthening of the Co−N(DMB) bond from

4

Cbls, such as MeCbl and AdoCbl, it has generally been

2.16 Å in MeCbl to 2.35 Å in NOCbl as determined by X-ray

−

65,16

described as a Co(III)−NO species.

crystallography.

The high degree of electron donation from

To test this assumption, we developed experimentally

validated electronic structure descriptions for NOCbl in its

base-on and base-oﬀ states. While our Abs spectrum of NOCbl

in ﬂuid solution at 300 K is similar to those reported in the

NO is particularly evident from an analysis of the NLMOs and

NBOs (Figure 10) derived from the canonical MOs obtained

with DFT. On the basis of this analysis, the doubly occupied

2

3d BD-NLMO contains 58% Co 3d orbital character (Table

z

2

5,2

literature,

we discovered a previously unobserved thermo-

5), consistent with a very covalent Co−NO bond. Anothe₊r

unique feature of the Co−NO bond in NOCbl and NOCbi

compared to the axial bonds in other Co(III) corrinoid species

is the presence of a sizable π-backbonding interaction involving

the Co 3d_y_zand NO π * orbitals. Inspection of the Co 3d

chromism for this species (Supporting Information, Figures

+

S3). From a comparison to the Abs data obtained for NOCbi ,

a model of base-oﬀ NOCbl, we conclude that an increase in

temperature from 4.5 to 300 K causes a shift in the base-on to

base-oﬀ equilibrium for NOCbl, favoring dissociation of the

N(DMB) ligand at high temperatures. This change in Co

coordination environment has, however, negligible eﬀects on

⊥

yz

BD-NLMO reveals that the NO ligand contribution to this

orbital is 16%, leading to an estimate for the extent of π

donation of ∼0.32 electrons. This backbonding partially

M

dx.doi.org/10.1021/ic500986x | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Table 6. Summary of NBO and NLMO Results for O Cbl. Energies, Occupancies, and Percent Co/O_O₂Compositions of the

2

Co-Based NBOs Are Shown for Alpha (Majority Spin) and Beta (Minority Spin) Orbitals, in Addition to the Percent

Contributions of These NBOs to the Corresponding NLMOs. Low-Occupancy NBOs, which Correspond to Formally Empty

a

Orbitals, Are Highlighted in Red

a

NA indicates values not availalable from NLMO composition analysis, but estimated to be close to 100%.

Table 7. Experimental and DFT-Computed Spin Hamiltonian Parameters for O Cbl. Hyperﬁne Coupling Constants Are Given

2

in Megahertz (MHz)

1

7

17

g values

A(Co)

A( O )

α

β

g₁

g₂

g₃

A₁

A₂

A₃

A₁

A₂

A₃

A₁

A₂

A₃

a

b

experiment

DFT

1.993

1.992

2.013

2.008

2.089

2.041

−30

−28

−64.8

−50.8

−18.7

−1.1

−167

−146

59

77

*

−201

−191

70

81

*

44

46

a

b

From ref 65. Values estimated to be between 0 and 20 MHz.

2

6

compensates for the large amount of σ donation from the NO

ligand into the Co 3d_zorbital, and represents an

Extension to O Cbl. Previous crystallographic and EPR

2

66

2

studies have indicated that Co(II)Cbl binds molecular oxygen

reversibly to yield superoxocobalamin (O Cbl) as the ﬁrst step

in the autoxidation process that eventually yields H

Cbl may also be of physiological importance as free

cobalamin is present predominantly in the Co(II)Cbl state in

vivo. Given the similar frontier orbitals of NO and O , which

has been widely exploited to mimic the binding of dioxygen to

metal centers in proteins by using nitric oxide, it is interesting

to compare the electronic structures of NOCbl and O Cbl.

While crystallographic data for these species have revealed that

both the NO and O molecules ligate to the Co center in a bent

end-on fashion, the axial Co−N(DMB) bond lengths diﬀer by

.29 Å between NOCbl (2.35 Å) and O Cbl (2.06 Å).

2

unprecedented mechanism by which the electronic properties

of the Co(III) ion can be modulated by the axial ligands.

The high degree of NO → Co σ donation in NOCbl

weakens the Co−N(DMB) bond to the point that at high

temperature, the base-on to base-oﬀ equilibrium favors a

unique pentacoordinate species in aqueous solution. In the case

2

+

OCbl .

2

O

2

67

2

+

of NOCbi , an eﬀectively ﬁve-coordinate species is present at

all temperatures, suggesting that the strong trans inﬂuence

exerted by the NO ligand precludes the binding of a weakly

donating water molecule even at 4.5 K. Nonetheless, during the

2

+

geometry optimization of a complete NOCbi model, the lower

16

26

0

water ligand did not fully dissociate. A comparison of this

structure and that obtained for the truncated model reveals

major diﬀerences in terms of the orientation of the water

molecule, indicating that the propionamide side chains of the

corrin ligand, which were absent in the truncated model, may

play an important role in modulating the lower axial bonding

interaction in cobalamins.

Hence, the Co−N(DMB) bond length in O Cbl is also

2

5

2

considerably shorter than it is in MeCbl (2.16 Å) but very

52

similar to that reported for CNCbl (2.041 Å), which is

considered to possess a moderately strong σ donor in the upper

axial position based on computational analyses and the fact that

4

this species exhibits a “typical” Co(III)Cbl Abs spectrum.

The NLMOs for O Cbl are shown in Figure 11, and the

compositions of the relevant NBOs and NLMOs are listed in

Table 6 (note that our electronic structure description for

O Cbl is supported by a previous single-crystal EPR character-

2

Further experimental support for our bonding description for

NOCbl is provided by published NMR data, which indicate

that the ¹⁵N-resonance of the NO ligand undergoes a 40 ppm

upﬁeld shift upon protonation and dissociation of the DMB

2

66

ization of this species, given the good agreement between the

11

ligand in the lower axial position. Although the observed

change in electron shielding experienced by the ¹⁵N nucleus

was originally attributed to an increase in the Co−N−O bond

angle from base-on to base-oﬀ NOCbl, our rR and DFT results

provide compelling evidence that the Co−NO core structures

of these two species are in fact virtually identical. Instead, our

computational data suggest that the enhanced shielding of the

computed and experimental g values and hyperﬁne coupling

constants, see Table 7). Because of the spin-unrestricted

formalism used in these calculations, the NLMOs have

occupancies of 1.00 and are divided into a set of spin-up (α,

majority spin. Figure 11, left) and spin-down orbitals (β,

minority spin. Figure 11, right). Inspection of the NLMOs of

O Cbl reveals that these orbitals are very similar in composition

to the NLMOs of NOCbl, although with some important

2

1

5

N nucleus in base-oﬀ NOCbl reﬂects the weaker σ-

antibonding interaction between Co and the lower axial ligand₊

in that species (cf. MOs #164 of NOCbl and #130 of NOCbi

in Figures 8 and 9, respectively), with the consequent increase

in N(O) natural charge by 4% (see Table 2).

diﬀerences. For both sets of spin orbitals, the Co 3d and Co

xz

2

3d

NLMOs of O Cbl are nonbonding with respect to the

x −y

2

ligand framework, in an analogous manner as the NLMOs of

NOCbl. Additionally, the compositions of the β NLMOs of

N

dx.doi.org/10.1021/ic500986x | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

O Cbl (Table 6, right) reﬂect the presence of a relatively

ACKNOWLEDGMENTS

This work was supported in part by the National Science

Foundation Grant Nos. MCB-0238530 (to T.C.B.) and CHE-

2

■

covalent Co−O bond, with a Co 3d orbital contribution to the

2

Co 3d (BD) β NLMO of 41%, as compared to 58% predicted

z

for NOCbl (see Table 5). However, the Co contribution to the

0

840494. I.G.P. was supported in part by Grant 5T32GM08349

2

z

spin-up counterpart (i.e., the Co 3d (BD) α NLMO) is merely

via the Biotechnology Training Grant at University of WI-

Madison. The authors thank Prof. Clark Landis for his

assistance with the NBO analysis.

2

4%. Thus, the net σ donation to the Co(III) ion from O in

2

O Cbl is smaller than it is from NO in NOCbl, which is

2

0

consistent with the greater oxidizing power of O (E = −0.16

2

0

68

V vs NHE) versus NO (E = −0.8 V NHE). Likewise, the

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Publishers: New York, 2000; Vol. 35.

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dx.doi.org/10.1021/ic500986x | Inorg. Chem. XXXX, XXX, XXX−XXX

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Spectral and electronic properties of nitrosylcobalamin

DOI: 10.1021/ic500986x

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Article abstract of DOI:10.1021/ic500986x

Full text of DOI:10.1021/ic500986x

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