Reductive coupling of nitrogen monoxide (?NO) facilitated by heme/copper complexes

1404 Inorg. Chem. 2010, 49, 1404–1419

DOI: 10.1021/ic901431r

Reductive Coupling of Nitrogen Monoxide (•NO) Facilitated by Heme/Copper

Complexes

Jun Wang,^†Mark P. Schopfer,^†Simona C. Puiu,^†Amy A. N. Sarjeant,^†and Kenneth D. Karlin*^,†,‡

^†Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218 and ^‡Department of

Bioinspired Science, Ewha Womans University, Seoul 120-750, Korea

Received July 21, 2009

The interactions of nitrogen monoxide (•NO; nitric oxide) with transition metal centers continue to be of great interest,

in part due to their importance in biochemical processes. Here, we describe •NO_(g)reductive coupling chemistry of

possible relevance to that process (i.e., nitric oxide reductase (NOR) biochemistry), which occurs at the heme/Cu

active site of cytochrome c oxidases (CcOs). In this report, heme/Cu/•NO_(g)activity is studied using 1:1 ratios of

heme and copper complex components, (F₈)Fe (F₈= tetrakis(2,6-difluorophenyl)porphyrinate(2-)) and

[(tmpa)Cu^I(MeCN)]^þ(TMPA = tris(2-pyridylmethyl)amine). The starting point for heme chemistry is the mononitrosyl

complex (F₈)Fe(NO) (λ_max= 399 (Soret), 541 nm in acetone). Variable-temperature ¹H and ²H NMR spectra reveal a

broad peak at δ = 6.05 ppm (pyrrole) at room temperature (RT), which gives rise to asymmetrically split pyrrole peaks

at 9.12 and 8.54 ppm at -80 ꢀC. A new heme dinitrosyl species, (F₈)Fe(NO)₂, obtained by bubbling (F₈)Fe(NO) with

•NO_(g)at -80 ꢀC, could be reversibly formed, as monitored by UV-vis (λ_max= 426 (Soret), 538 nm in acetone), EPR

(silent), and NMR spectroscopies; that is, the mono-NO complex was regenerated upon warming to RT. (F₈)Fe-

(NO)₂reacts with [(tmpa)Cu^I(MeCN)]^þand 2 equiv of acid to give [(F₈)Fe^III]^þ, [(tmpa)Cu^II(solvent)]^2þ, and N₂O_(g),

fitting the stoichiometric •NO_(g)reductive coupling reaction: 2•NO_(g)þ Fe^IIþ Cu^Iþ 2H^þf N₂O_(g)þ Fe^IIIþ Cu^IIþ

H₂O, equivalent to one enzyme turnover. Control reaction chemistry shows that both iron and copper centers are

required for the NOR-type chemistry observed and that, if acid is not present, half the •NO is trapped as a (F₈)Fe(NO)

complex, while the remaining nitrogen monoxide undergoes copper complex promoted disproportionation chemistry.

As part of this study, [(F₈)Fe^III]SbF₆was synthesized and characterized by X-ray crystallography, along with EPR

(77 K: g = 5.84 and 6.12 in CH₂Cl₂and THF, respectively) and variable-temperature NMR spectroscopies. These

structural and physical properties suggest that at RT this complex consists of an admixture of high and intermediate

spin states.

Introduction

site, a heme with a proximate nonheme iron metal center.^2-4

Actually, these two enzyme classes are evolutionarily

related.^5,6The heme/M (M = Fe or Cu) centers both possess

a number of conserved histidines: one is a proximal ligand

for the heme of the binuclear heme/M center (as in hemo-

globin/myoglobin), and three other histidine imidazole

donors bind to the so-called Cu_B(in CcOs) and Fe_B(in

NORs). NORs are found in anaerobic denitrifiers which,

instead of using dioxygen to pass electrons obtained from

metabolic processes, use nitrogen oxides. The following

In this report, we describe the reductive coupling of 2 mol

equiv of nitrogen monoxide (•NO_(g); nitric oxide) to nitrous

oxide (N₂O_(g)), 2•NO_(g)þ 2 H^þþ 2 e^-f N₂O_(g)þ H₂O,

mediated by a heme plus copper complex in the presence of

acid. The particular compounds employed and the overall

reaction discussed are shown in Scheme 1. This follows our

previous paper¹on similar chemistry carried out by a bi-

nuclear heme/Cu assembly possessing a binucleating ligand.

That was the first example of such chemistry with heme/Cu,

modeling the heterobinuclear center present at the active site

of cytochrome c oxidase (CcO).

-

biochemical pathway comes into play: NO₃f NO₂

f

•NO f N₂O f N₂.^4,7Each reaction step is catalyzed by

metalloproteins.

In fact, bacterial •NO reductases (NORs) effect this

reductive coupling of •NO, and these have a related active

€

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*To whom correspondence should be addressed. E-mail: karlin@jhu.edu.

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r

pubs.acs.org/IC

Published on Web 12/23/2009

2009 American Chemical Society

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1405

Scheme 1

metal centers, influencing the regulation of mitochondrial

reactive oxygen species generation and preventing cyto-

chrome c release. There are also literature discussions con-

cerning the reaction of •NO_(g)with CcO intermediates

leading to its oxidation to nitrite;²³this process may be

biologically important for the purpose of generating pools

of the latter, which may later be reduced back to NO(perhaps

also by reduced CcO).^18,24The interplay of •NO and O₂

chemistry at CcO metal centers and the control of relative

levels of •NO (and nitrite) and O₂(and rates of respiration)

are biologically critical processes.

Thus, the reductive coupling of nitric oxide is of chemical

and biological interest, and the study of systems undergoing

this chemistry is part of our larger research program in the

investigation of small molecule gases (O₂, NO, and CO) and

their interactions with heme/M (M = Fe or Cu) binuclear

assemblies.^1,10,25-27As stated, here, we report on a system

employing heme/Cu complexes, in fact, component heme

and copper complexes along with the presence of acid, which

function together to facilitate the stoichiometric •NO_(g)

coupling reduction to N₂O_(g), Scheme 1. As part of the

description and chemistry involved, we describe the genera-

tion and some of the physical properties of a new complex,

a heme dinitrosyl species (F₈)Fe(NO)₂. Also, the X-ray

structure and spectroscopic characterization of a new

heme-iron(III) complex, [(F₈)Fe^III](SbF₆) (F₈= tetrakis-

(2,6-difluorophenyl)porphyrinate(2-)) are reported.

In the penultimate step in the mitrochondrial electron-

transport chain, CcO’s catalyze the four-electron reduction

of dioxygen to water; this couples to inner-membrane proton

translocation and a proton/charge gradient which powers the

subsequent synthesis of ATP.^8-10However, certain types of

CcOs, such as ba₃and caa₃oxidases from Thermus thermo-

philus, cbb₃oxidase from Pseudomonas stutzeri, and bo₃from

Escherichia coli also carry out nitrogen monoxide reductive

coupling, that is, NOR chemistry,¹¹and this subject has

recently received considerable attention from the biophysical

and computational research communities.^2,11-14Lu and

co-workers¹⁵have investigated such chemistry coming from

a designed/modified small protein (myoglobin) model sys-

tem. Here, a Cu_Bcenter was designed and integrated into a

wild-type myoglobin, leading to the formation of a protein

with a binuclear heme/Cu center, which with an added

external reductant was able to catalyze the •NO_(g)to N₂O

conversion. Yet, we are far from having a sufficient under-

standing of the underlying metal/•NO_(g)and metal ion

mediated NO-NO coupling chemistry.

Experimental Section

Materials and Methods. Unless otherwise stated, all solvents

and chemicals used were of commercially available analytical

grade. Nitrogen monoxide (•NO) gas was obtained from Math-

eson Gases and passed multiple times through a column con-

taining KOH pellets and through a liquid N₂cooled trap to

remove impurities (see also the Supporting Information of

refs 27 & 28). The purified •NO_(g)was passed into a Schlenk flask

placed in liquid N₂, to freeze. For use in reactions, this frozen gas

was briefly warmed with the acetone/dry ice bath (-78 ꢀC) and

allowed to pass into an evacuated Schlenk flask (typically

50 mL) fitted with a septum. The addition of •NO_(g)to metal

complex solutions was effected by transfer via a three-way long

syringe needle. Dinitrogen oxide (N₂O) gas was purchased from

Airgas as a custom mixture, at a concentration of 250 ppm,

balanced with dinitrogen at 1 atm. Dichloromethane (CH₂Cl₂;

DCM), acetonitrile (MeCN), methanol (MeOH), and pentane

were used after passing through a 60-cm-long column of acti-

vated alumina (Innovative Technologies, Inc.) under argon.

Tetrahydrofuran (THF) and acetone were purified and dried

by distillation from sodium/benzophenone ketyl. Preparation

and handling of air-sensitive compounds were performed under

an argon atmosphere using standard Schlenk techniques or in an

MBraun Labmaster 130 inert atmosphere (<1 ppm O₂,

<1 ppm H₂O) drybox filled with nitrogen gas. Deoxygenation

In addition, the interaction of •NO_(g)with CcO’s is of

considerable importance.^16-22Nitrogen monoxide reversibly

inhibits mitochondrial respiration by binding to the CcO

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1406 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

of the solvents was effected by either repeated freeze/pump/

thaw cycles or bubbling with argon for 30-45 min.

30 min, and degassed heptane was added until a solid product

precipitated. The brown-red solid obtained (60 mg, 77%) was

filtered and dried under a vacuum and then stored in the drybox.

UV-vis (λ_max, nm): THF, 410 (Soret), 546. IR (cm^-1): ν_NO1670

in THF. EPR spectrum, see Results and Discussion. Anal. Calcd

UV-vis spectra were recorded on a Hewlett-Packard Model

8453A diode array spectrophotometer equipped with

a

two-window quartz H.S. Martin Dewar filled with cold MeOH

(þ25 ꢀC to -85 ꢀC) maintained and controlled by a Neslab

VLT-95 low-temperature circulator. Spectrophotometer cells

used were made by Quark Glass with a column and pressure/

vacuum side stopcock and 1 cm path length. Electron para-

magnetic resonance (EPR) spectra were recorded on a Bruker

EMX spectrometer controlled with a Bruker ER 041 X G

microwave bridge operating at the X-band (∼9.4 GHz). ESI

mass spectra were acquired using a Finnigan LCQDeca ion-trap

mass spectrometer equipped with an electrospray ionization

source (Thermo Finnigan, San Jose, CA). The heated capillary

temperature was 250 ꢀC, and the spray voltage was 5 kV. Gas

chromatography analysis was performed on a Varian CP-3800

instrument equipped with a 1041 manual injector, electron

for {(THF)(F₈)Fe(NO) THF}, C₅₂H₃₆F₈FeN₅O₃: C, 63.30; H,

3

3.68; N, 7.10. Found: C, 62.76; H, 3.60; N, 6.94.

Synthesis of [(F₈)Fe^III]SbF₆. This synthesis is a modified

version of that reported earlier for tetraphenylporphyrin.³⁵In

a glovebox filled with N₂, (F₈)FeCl (0.30 g, 0.354 mmol) and

AgSbF₆(0.128 g, 0.373 mmol) were weighed and transferred to a

Schlenk flask equipped with a stir bar. The solid materials were

dissolved in 35 mL of freshly distilled THF, and the solution was

stirred at room temperature for 1 h under reduced light. The

reaction mixture was then filtered to remove AgCl. The filtrate

was dried in vacuo, redissolved in 7 mL of CH₂Cl₂, and

precipitated twice with the addition of 80 mL of pentane,

yielding a purple powder (250 mg, 83%). UV-vis (λ_max, nm):

CH₂Cl₂, 328, 394 (Soret), 512; THF, 328, 394 (Soret), 510. IR

(Nujol, cm^-1): ν_SbF6657 cm^-1. EPR spectrum at 77 K: g = 5.84

(in DCM); g = 6.12 (in THF). ESI-MS: 812.5, (F₈)Fe. ¹H NMR

(CD₂Cl₂): δ 27.2 and 11.6 (8 pyrrole), 10.2 (4 para), 9.4 (8 meta).

¹H NMR (THF-d₈): δ 54.7 and 14.8 (8 pyrrole), 10.4 (4 para),

˚

conductivity detector, and a 25 m 5 A molecular sieve capillary

column. Ion chromatography analysis (ICA) was performed on

a Dionex DX-120 Ion chromatograph, with an AS40 automated

sampler, and an IonPac AS14 (4*250 mm) column. The eluent

was 3.5 mM Na₂CO₃along with 1.0 mM NaHCO₃. See below

for the procedure for generating a sample for ICA. Infrared

spectra (IR) were recorded on a Bruker Vector 22 instrument

controlled by OPUS-NT software at room temperature.

9.3 (8 meta). Anal. Calcd for {(F₈)FeSbF₆1.5CH₂Cl₂},

C

3

_45.5H₂₃Cl₃F₁₄FeN₄Sb: C, 46.48; H, 1.97; N, 4.75. Found: C,

46.38; H, 2.12; N, 4.66. Crystals, suitable for X-ray diffraction,

were obtained by layering a concentrated tetrahyrdrofuran

solution of [(F₈)Fe^III]SbF₆with pentane. The purple-colored

X-ray-quality crystals are formulated as [(F₈)Fe^III(THF)₂]SbF₆.

See the Results and Discussion for further information.

Low-Temperature NMR Spectroscopic Measurements. Multi-

2

nuclear (¹H and H) NMR spectroscopic measurements were

performed at various temperatures under a N₂atmosphere. ¹H

NMR spectra were measured on a Bruker 400 MHz spectro-

meter, while ²H NMR spectra were measured on a Varian

Mercury 500 MHz spectrometer. Chemical shifts were reported

as δ values relative to an internal standard of the deuterated

solvent being used. Measurement of spectra for the mono- and

dinitrosyl heme complexes was carried out in septum-capped

NMR tubes, and •NO_(g)was added via an airtight syringe to

-78 ꢀC solutions.

Synthesis of [(tmpa)Cu^II(MeCN)](ClO₄)₂. TMPA (1.51 g,

5.20 mmol) and Cu(ClO₄)₂6H₂O (1.93 g, 5.21 mmol) were

3

dissolved in a total of 40 mL of CH₃CN and allowed to stir for

30 min, whereupon a dark blue solution developed. Diethyl

ether (90 mL) was added to give a crude precipitate. Recrystal-

lization of this solid from CH₃CN/ether and drying in vacuo

gave a light blue powder in a yield of 86% (2.66 g). Anal. Calcd

for [(tmpa)Cu^II(MeCN)](ClO₄)₂, C₂₀H₂₁Cl₂CuN₅O₈: C, 40.45;

H, 3.56; N, 11.79. Found: C, 40.55; H, 3.53; N, 11.80. UV-vis

(CH₃CN, λ_max, nm (ε, M^-1cm^-1)): 255 (14 700), 855 (254). EPR

spectrum in acetone at 77 K (see Figure S1, Supporting In-

formation): g_^= 2.22, g = 2.02, A_^= 105 G, A = 71 G.

Synthesis of [(tmpa)Cu^II(NO₂)]PF₆. To a 100 mL Schlenk

flask equipped with a magnetic stir bar was added 200 mg of

[(tmpa)Cu^I(MeCN)]PF₆in degassed CH₃CN/THF (50:50) un-

der a N₂atmosphere. The reaction flask was incubated in an

acetone/dry ice bath. Excess •NO_(g)was bubbled through this

solution, and the reaction mixture was allowed to stir for 30 min,

with the color turning from yellow to purple. The solution was

warmed to room temperature (RT), with its color turning to

green, and this was kept stirring for 2 h. Solvents were removed

under reduced pressure, and then recrystallization of the green

solid from CH₃CN/ether gave a green microcrystalline solid

(75%). IR (in DCM, cm^-1): ν_as(NO₂) 1390, ν_as(NO₂) 1330.

UV-vis (λ_max, nm, in CH₂Cl₂): 301, 415 (see Figure S2, Sup-

porting Information). EPR spectrum in acetone at 77 K (see

Figure S3, Supporting Information): g_^= 2.21, g = 2.01, A_^=

84 G, A = 80 G. Anal. Calcd for [(tmpa)Cu^II(NO₂)]PF₆,

C₁₈H₁₈CuF₆N₅O₂P: C, 39.68; H, 3.33; N, 12.85. Found: C,

39.76; H, 3.50; N, 12.27. Nitrite (NO₂^-) was the only detectable

ion in the green solid, as determined using ICA. See below for

the procedure for generating a sample for ICA.

X-Ray Structure Determination. X-ray diffraction was per-

formed at the facility of the chemistry department of Johns

Hopkins University. The X-ray intensity data were measured on

an Oxford Diffraction Xcalibur3 system equipped with a gra-

phite monochromator and an Enhance (Mo) X-ray Source

˚

(λ = 0.71073 A) operated at 2 kW power (50 kV, 40 mA) and

a CCD detector. The frames were integrated, scaled, and

corrected for absorption using the Oxford Diffraction CrysA-

lisPRO software package.

Syntheses. (F₈)FeCl (F₈= tetrakis(2,6-difluorophenyl)por-

phyrinate(2-)),²⁹(F₈-d₈)FeCl,³⁰(F₈)Fe^II,³⁰(F₈-d₈)Fe^II,³⁰

(F₈)Fe(NO),¹tris(2-pyridylmethyl)amine (TMPA),³¹[(tmpa)-

Cu^I(MeCN)]PF₆,³¹[(tmpa)Cu^I(MeCN)]B(C₆F₅)₄,³²[Cu^I(MeCN)₄]-

SbF₆,³³and [H(C₂H₅OC₂H₅)₂][B(C₆F₅)₄] (HBArF)³⁴were pre-

pared from literature reports.

Synthesis of (THF)(F₈)Fe(NO). A solution of 75 mg (0.092

mmol) of (F₈)Fe^IIin 5 mL of THF was cooled to -78 ꢀC using a

dry ice/acetone bath. Under Ar, 20 mL of •NO_(g)at ∼1 atm was

added using a gastight syringe. The solution was stirred for

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Synthesis of [(tmpa)Cu^I(MeCN)]SbF₆. This synthesis fol-

lowed a procedure similar to that reported earlier for

[(tmpa)Cu^I(MeCN)₄]PF₆.³¹A solution of 200 mg (0.689 mmol)

of TMPA ligand in 10 mL of distilled CH₃CN was added to

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Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1407

319 mg (0.689 mmol) of [Cu(CH₃CN)₄]SbF₆in a 100 mL

Schlenk flask in the glovebox; the solution was stirred for 1 h.

Afterward, 75 mL of diethyl ether was added to the bright

orange solution until a slight cloudiness was observed to devel-

op. The solution was filtered through a medium-porosity frit,

and the compound obtained was recrystallized from CH₃CN/

ether. The precipitate formed was dried under vacuum, leading

to a yellow-orange solid (78%). ¹H NMR (CD₃CN): δ 8.93

(br, 3H), 7.83 (m, 3 H), 7.50 (br, 6 H), 4.3 (v br, 6 H), 2.01 (s, 3 H,

CH₃CN).

Synthesis of [(tmpa)Cu^II(NO₂)]SbF₆. To a 100 mL Schlenk

flask equipped with a magnetic stir bar was added 300 mg of

[(tmpa)Cu^I(MeCN)]SbF₆in degassed CH₃CN/THF (50:50)

under a N₂atmosphere. The reaction flask was then incubated

in an acetone/dry ice bath and the solution bubbled with excess

NO_(g). The reaction mixture was allowed to stir for 30 min with

the color turning from yellow to purple. Subsequently, the

closed system warmed to RT, now turning to green, and

this solution was stirred at RT for 2 h. Solvents were re-

moved under reduced pressure, and recrystallization of the

resulting solid from CH₃CN/ether gave a green microcrystalline

solid [(tmpa)Cu^II(NO₂)]SbF₆(70% yield). IR (in CH₂Cl₂,

cm^-1): ν_as(NO₂) 1390, ν_as(NO₂) 1330. Anal. Calcd for

[(tmpa)Cu^II(NO₂)]SbF₆, C₁₈H₁₈CuF₆N₅O₂Sb: C, 34.01; H, 2.85;

N, 11.02. Found: C, 33.72; H, 2.80; N, 10.58. UV-vis (λ_max, nm, in

CH₂Cl₂): 301, 415. Nitrite (NO₂^-) was the only ion detected

following ion chromatography analysis on this green solid. See

below for the procedure for generating a sample for ICA.

S4, Supporting Information). On the basis of the known extinc-

tion coefficients for (THF)(F₈)Fe(NO) {λ_max= 410 nm (Soret),

ε = 176 000 M^-1cm^-1; 546 nm, ε = 8300 M^-1cm^-1}, the

mononitrosyl species was formed in approximately 90% yield

on the basis of the reaction, (F₈)Fe(NO)₂þ (F₈)Fe^IIf 2(THF)-

(F₈)Fe(NO) (in THF solution).

Reaction of (F₈)Fe(NO)₂with [(tmpa)Cu(MeCN)]PF₆and

Acid. Complex (F₈)Fe(NO) (0.050 g, 0.059 mmol) was dissolved

in 10 mL of distilled acetone in a 50 mL Schlenk flask and then

cooled to -78 ꢀC using an acetone/dry ice bath. Excess •NO_(g)

was bubbled through the orange cold solution using a three-way

syringe, and then the excess •NO_(g)was removed via vacuum/

purge cycles. After stirring for 30 min, the orange solution

was added to the prechilled [(tmpa)Cu^I(MeCN)][B(C₆F₅)₄] in

10 mL of an acetone/MeCN (1:1; 0.064 mg, 0.060 mmol)

solution via a two-way syringe needle. After stirring for

10 min, a 5 mL acetonitrile solution of [H(C₂H₅OC₂-

H₅)₂][B(C₆F₅)₄] (HBArF) (0.098 mg, 0.118 mmol) was added

to the cold orange solution mixture. It turned to brown upon

warming to RT. The solution was concentrated in vacuo, and

addition of deoxygenated pentane led to a brown solid. UV-vis

(λ_max, nm): CH₂Cl₂, 394 (Soret), 512; THF, 394 (Soret), 511. IR

(Nujol, cm^-1): no related ν_NOobserved. EPR spectrum in

acetone (77 K): g = 6.12 (heme-Fe^III); g_^= 2.21, g = 2.01,

A_^= 100 G, A = 68 G (Cu^II). ESI-MS in MeCN: (813, (F₈)-

Fe þ H; 853, (F₈)Fe þ MeCN; 871, (F₈)Fe þ H₂O þ MeCN;

353, (tmpa)Cu). A 10 mL CH₂Cl₂solution of the brown solid

product was mixed with 10 mL of aqueous NaCl solution

(400 uM) and stirred for half an hour. Ion chromatography

analysis of this upper aqueous layer extract gave no indication

for the presence of any nitrite ion (NO₂^-).

Generation of (F₈)Fe(NO)₂. A 5 mL distilled acetone solution

of (F₈)Fe(NO) (5 ꢀ 10^-6mol/L) was taken in a UV-vis cuvette

assembly under argon; it was cooled to -78 ꢀC, and an initial

spectrum was recorded. Excess •NO_(g)was bubbled through the

cold solution using a three-way needle syringe, and the new

spectrum (of (F₈)Fe(NO)₂) was recorded, λ_max= 426 (Soret),

538 nm. Additional bubbling with •NO_(g)did not change the

spectrum further. This resulting species (F₈)Fe(NO)₂released

NO to form (F₈)Fe(NO) upon warming to RT, and

(F₈)Fe(NO)₂was regenerated when cooling to -80 ꢀC or adding

more NO_(g). The other protocol for (F₈)Fe(NO)₂generation was

bubbling excess •NO_(g)into the reduced complex (F₈)Fe^IIat

-80 ꢀC, while (F₈)FeNO was formed if •NO_(g)reacted with

(F₈)Fe^IIat RT. ¹H NMR (CD₂Cl₂): δ 9.13 and 8.93 (8 pyrrole),

7.84 (4 para), 7.45 (8 meta). EPR spectrum in acetone at 77 K:

silent. The related deuterated complex, (F₈-d₈)Fe(NO)₂, was

generated in a similar manner, except starting with (F₈-d₈)Fe-

(NO). (F₈-d₈)Fe(NO) was synthesized from (F₈-d₈)Fe^IInitrosy-

lation by bubbling •NO_(g)through the (F₈-d₈)Fe^IIsolution

(in CH₂Cl₂) and removal of excess •NO_(g)and solvents to yield

a purple solid.

Titration of (F₈)Fe(NO)₂with (F₈)Fe^II. (F₈)Fe^II(0.0019 g,

0.0023 mmol) was dissolved in 100 mL of deaerated THF inside

the glovebox. From the red solution, 10 mL was transferred into

a Schlenk flask, and under an Ar atmosphere on the benchtop

this was cooled to -78 ꢀC and a spectrum recorded {λ_max= 422

(Soret), 542 nm}. Afterward, excess •NO_(g)was bubbled

through this cold solution using a three-way long needle syringe,

the resulting new spectrum of the orange product (F₈)Fe(NO)₂

was recorded, {λ_max= 410 (Soret), 540 nm}. Additional bub-

bling with •NO_(g)did not change the spectrum further. The cold

mixture was then purged with an Ar flow for 1 min, and the

entire flask was evacuated and refilled with Ar three times to

remove the excess •NO_(g). There was no spectral change upon

excess •NO_(g)removal. Approximately 1 equiv of (F₈)Fe^II

was added to the cold solution by transferring 10 mL of the

cold (-78 ꢀC) (F₈)Fe^IIstock solution in THF with a cannula

technique. At -78 ꢀC, the spectrum indicated it was a

mixture of mononitrosyl, dinitrosyl, and (F₈)Fe^II. However,

upon warming to RT, only one product was present,

(THF)(F₈)Fe(NO), {λ_max= 410 (Soret), 546 nm} (see Figure

Gas chromatography analysis of the head space of this

reaction mixture reveals N₂O_(g)formed in a yield of 84%

according to the stoichiometry 2•NO þ 2 e^-þ 2 H^þf N₂O_(g)

þ H₂O. Thus, the metal complex products of the reaction

are [(F₈)Fe^{III þ}and [(tmpa)Cu^II(solvent)]^2þ, along with N₂O_(g)

] .

See the Results and Discussion for further information or

clarification.

Reaction of (F₈)Fe(NO)₂with [(tmpa)Cu(MeCN)]PF₆. Com-

plex (F₈)Fe(NO) (0.040 g, 0.047 mmol) was dissolved in 10 mL

of distilled acetone and then cooled to -78 ꢀC using an acetone/

dry ice bath. Excess •NO_(g)was bubbled through the cold

solution using a three-way syringe. After stirring for 30 min,

the orange solution of (F₈)Fe(NO)₂was added to a precooled

[(tmpa)Cu^I(MeCN)]PF₆(0.026 mg, 0.048 mmol) solution in

10 mL of acetone/MeCN (1:1) via a two-way syringe needle,

and the mixture was allowed to stir for 30 min. After warming to

RT, the solution mixture kept stirring for 1 h and then was

concentrated in a vacuum. The addition of 70 mL of deoxyge-

nated pentane precipitated the orange product solution as a

mixture of purple and green solids. An EPR spectrum indicated

that the product was a mixture of (F₈)Fe(NO) and Cu^IIspecies,

while an IR spectrum of this solid (Nujol mull) indicated that

one •NO molecule is still bound to the (F₈)Fe fragment, ν_NO

=

1684 cm^-1. UV-vis (λ_max, nm): THF, 410 (Soret), 546; CH₂Cl₂,

400 (Soret), 540. To determine what other nitrogen oxide

products formed, a series of stock solutions of synthetically

derived [(tmpa)Cu(NO₂)]SbF₆(vide supra) were generated,

mixing (30 min) 10 mL of CH₂Cl₂with 10 mL of aqueous

NaCl (160 μM). The upper aqueous layer extract was taken for

nitrite ion analysis on the Dionex DX-120 Ion chromatograph

(see above for the procedure for generating a sample for ICA),

and then a calibration curved was formed. Afterward, the

product mixture of the reaction of 20 mg (F₈)Fe(NO)₂þ 13

mg [(tmpa)Cu^I(MeCN)]PF₆(1:1 molar ratio) was analyzed for

ion fractions via the same method. Via this nitrite analysis,

[(tmpa)Cu(NO₂)]PF₆was found to form in a yield of 95%, on

the basis of the reaction stoichiometry: 3•NO þ [(L)Cu^I]^þ

f

N₂O þ [(L)Cu^II(NO₂^-)]^þ. Thus, the metal complex products of

1408 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

Scheme 2

which have been characterized and will be discussed in this

report.

As shown in Scheme 3, the mononitrosyl complex

(F₈)Fe(NO) reacted with •NO_(g)to form the dinitrosyl

species, (F₈)Fe(NO)₂, very similar in properties to the dini-

trosyl complex generated in situ in the ⁶L system,

(⁶L)Fe(NO)₂(Scheme 2).¹Once discovering that such com-

plexes exist and can be generated in a stoichiometric manner

(vida infra), we wished to take advantage of this fact that

exactly two •NO molecules are present for each heme or

6

heme/Cu assembly. This would, as it turns out for the L

binucleating system and also here, allow for a stoichiometric

reaction, for example, one synthetic model enzyme turnover:

2•NO þ Fe^IIþ Cu^Iþ 2H^þf N₂O_ðgÞþ Fe^IIIþ Cu^IIþ H₂O

ð1Þ

Exactly this reaction occurs when (F₈)Fe(NO)₂is added to

[(tmpa)Cu^I(MeCN)]^þin the presence of 2 equiv of acid, the

latter in the form of H(Et₂O)₂[B(C₆F₅)₄]. The NOR type

reaction occurs and the particular products obtained are

[(F₈)Fe^III]^þ, [(tmpa)Cu^II(solvent)]^2þ, and N₂O_(g). However,

when acid is absent, the reaction of (F₈)Fe(NO)₂and

[(tmpa)Cu^I(MeCN)]^þyields different products, and different

•NO_(g)/metal chemistry takes place (Scheme 3). Details are

provided in the subsequent sections.

Mononitrosyl Complexes (F₈)Fe(NO) and (THF)-

(F₈)Fe(NO). We recently¹reported on the synthesis and

X-ray structure of (F₈)Fe(NO), that formed via the

reductive nitrosylation of (F₈)Fe^IIICl. It can also be

generated by bubbling •NO_(g)through a (F₈)Fe^IIsolu-

tion, that is, (F₈)Fe^IIþ NO f (F₈)Fe(NO) (see the

Experimental Section). (F₈)Fe(NO) is a stable complex

as a solid and in solution, and unreactive toward dioxy-

gen. It displays a N-O stretching frequency at 1684 cm^-1

(Nujol), 1691 cm^-1in CH₂Cl₂solution, and 1670 cm^-1in

THF. It gives the expected classic rhombic EPR spectrum

with three-line hyperfine splitting pattern due to unpaired

electron interaction with ¹⁴N (I = 1) of the nitrosyl

ligand (g₁= 2.113, g₂= 2.078, g₃= 2.025), Figure 1.

In the course of our studies with heme/Cu/O₂reactivity

using (F₈)Fe^II, we have explored the use of various

solvents and found, for example, that acetone, THF,

and RCN (R = Me, Et) solvents bind the heme as axial

“base” ligands.³⁰THF is particularly strong compared to

the others. In fact, THF is found to bind quite strongly

to (F₈)Fe(NO), affording (THF)(F₈)Fe(NO). We were

able to isolate this six-coordinate nitrosyl complex, the

first with an oxygen donor to be characterized by ele-

mental analysis, IR, EPR, and UV-vis spectroscopies.

In Table 1, we compare IR, EPR, and UV-vis para-

meters for (THF)(F₈)Fe(NO), (F₈)Fe(NO), various

other (porphyrinate)Fe(NO) complexes, and a previously

described bis-THF adduct, (THF)₂Fe^II.³⁸Figure 2 shows

the EPR spectrum of (THF)(F₈)Fe(NO) recorded at 77 K

in THF.

the reaction are (F₈)Fe(NO) plus a mixture of 1/3[(tmpa)Cu^II-

(NO₂)]PF₆and unreacted 2/3[(tmpa)Cu^I(solvent)]^þ. Gas

chromatography analysis of the head space of the original

reaction mixture reveals N₂O_(g)formed in a yield of 88%

according to this stoichiometry. See the Results and Discussion

for further information or clarification.

Results and Discussion

We previously reported on •NO_(g)reductive coupling

chemistry carried out by a binuclear heme/Cu assembly with

a binucleating ligand referred to as ⁶L; with the formation of

a heme-dinitrosyl complex (⁶L)Fe(NO)₂, the addition of a

copper(I) ion source along with acid led to excellent yields of

N₂O_(g)(Scheme 2).¹In that study, the use of a heterobinu-

cleating ligand made sense in terms of a synthetic strategy in

carrying out such cooperative heme/Cu/small-molecule in-

vestigations. This would vastly increase the liklihood that

this reductive coupling chemistry was not resulting from

heme-heme or copper-copper reactions.

In fact, the use of component mixtures of heme and

separate copper complexes has been previously very success-

ful for our study of heme/Cu/O₂reactivity, leading to the

generation and characterization of quite a large series of

heme/Cu/O₂adducts, including those with ⁶L.^10,25,36,37Thus,

here we chose to also apply that approach with respect to

•NO_(g)reductive coupling chemistry, thus hopefully effected

by a distinct heme together with a copper complex. Scheme 3

summarizes the chemical transformations described in this

paper and depicts all of the important individual complexes

The subfield of heme-nitrosyl complexes, synthetic or

biological, is already very mature, although it continues

ꢀ

(36) Chufan, E. E.; Puiu, S. C.; Karlin, K. D. Acc. Chem. Res. 2007, 40,

563–572.

(37) Ghiladi, R. A.; Ju, T. D.; Lee, D.-H.; Moenne-Loccoz, P.; Kaderli,

(38) Thompson, D. W.; Kretzer, R. M.; Lebeau, E. L.; Scaltrito, D. V.;

Ghiladi, R. A.; Lam, K.-C.; Rheingold, A. L.; Karlin, K. D.; Meyer, G. J.

Inorg, Chem. 2003, 42, 5211–5218.

€

S.; Neuhold, Y.-M.; Zuberbuhler, A. D.; Woods, A. S.; Cotter, R. J.; Karlin,

K. D. J. Am. Chem. Soc. 1999, 121, 9885–9886.

(39) Wyllie, G. R. A.; Scheidt, W. R. Chem. Rev. 2002, 102, 1067–1089.

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1409

Scheme 3

to draw considerable interest, due to more recently des-

cribed biological processes which utilize or process •NO

and its oxidized and reduced derivatives. There are many

spectroscopically and structurally characterized five-co-

ordinate and six-coordinate heme-nitrosyl complexes,

so-called {FeNO}⁷derivatives, the value of seven derived

from the six d electrons (i.e., 3d⁶) of iron(II) plus the

unpaired electron from •NO.^44,45It seems to be the case

that there are only quite small differences in structural

coordination parameters comparing complexes with dif-

ferent coordination numbers. However, IR spectra

clearly suggest that, with an axial “base” binding to a

trans/sixth position of a heme-NO moiety, the N-O

stretching frequency shifts to a lower value.⁴⁶For exam-

ple, (TPP)Fe(NO) has a N-O stretch at 1670 cm^-1, which

shifts to 1653 and 1625 when 4-methylpiperidine

(4-MePip) and 1-methylimidazole (1-MeIm) are axially

bound, respectively (Table 1). In the case of (F₈)Fe(NO)

(ν_(N-O)= 1684 cm^-1), ν_(N-O)decreases by only 15 cm^-1

with THF binding, but by 60 cm^-1with 1-MeIm bind-

ing.⁴⁰Thus, our data clearly show that THF is a signifi-

cant base, but much weaker than N-donor ligands such as

1-methylimidazole, which has been reported⁴⁰to effect a

much larger (60 cm^-1) shift to lower frequency.

Another difference between five- and six-coordinated

heme-NO species is their EPR spectroscopic behavior.

Five-coordinated complexes always exhibit the triplet

hyperfine as described above for (F₈)Fe(NO).^41,47De-

pending on the sixth axial trans ligand, (L)-

(porpyrinate)Fe(NO) complexes reveal two different

types of hyperfine splitting patterns. Most possess a

nine-line pattern localized to the g₂or g₃region,^41,48

due to the N hyperfine spitting of both the trans-N

ligand and •NO; that is, a triplet of triplets is observed.

There are only a few six-coordinate complexes with

O-donor trans ligands.^41,42(THF)(PPDME)Fe(NO)

shows a three-line spectrum, but with smaller g

values than that of five-coordinated(PPDME)Fe(NO)

(Table 1). It was suggested that this indicates electronic

interaction of THF occurs though the lone pair elec-

trons on the oxygen atom. Of course, this conclusion is

readily observed from IR spectroscopic data on our

own (THF)(F₈)Fe(NO) complex (vide supra). Also, an

EPR spectrum of this complex is nearly identical to

that observed for (THF)(PPDME)Fe(NO), with a

three-line pattern with g values (g₁= 2.100, g₂=

2.063, g₃= 2.011) altered somewhat from that seen

for (F₈)Fe(NO), see Figure 2 and Table 1.

Dinitrosyl Complex (F₈)Fe(NO)₂. This complex can be

generated by bubbling excess •NO_(g)through solutions of

either (a) the (F₈)Fe^IIreduced compound or (b) the

mononitrosyl complex (F₈)Fe(NO) (see the Experimental

Section). For purposes of characterization and reactivity

studies, excess •NO_(g)was removed by the application of

vacuum/purge cycles. An EPR spectrum of such a sample

of (F₈)Fe(NO)₂in acetone (77 K) shows the compound to

be EPR-silent (Figure 1). This is consistent with it being

diamagnetic, as might be expected by its formation from

(40) Praneeth, V. K. K.; Nather, C.; Peters, G.; Lehnert, N. Inorg. Chem.

2006, 45, 2795–2811.

(41) Cheng, L.; Richter-Addo, G. B. Binding and Activation of Nitric

Oxide by Metalloporphyrins and Heme. In Porphyrin Handbook; Kadish,

K. M., Simith, K. M., Guilard, R., Eds.; Academic Press: San Diego, CA, 2000;

Vol. 4, Chapter 33, pp 219-291.

(42) Yoshimura, T. Inorg. Chim. Acta 1982, 57, 99–105.

(43) Lorkovic, I.; Ford, P. C. J. Am. Chem. Soc. 2000, 122, 6516–6517.

(44) Enemark, J. H.; Feltham, R. D. Coord. Chem. Rev. 1974, 13, 339–

406.

(45) Lee, D.-H.; Mondal, B.; Karlin, K. D. NO and N₂O Binding and

Reduction. In Activation of Small Molecules: Organometallic and Bioinor-

ganic Perspectives; Tolman, W. B., Ed.; Wiley-VCH: New York, 2006;

pp 43-79.

(46) As is well understood, the sixth ligand provides additional electron

density to the iron ion, which leads to enhanced backbonding to the π*(NO)

orbital, thus weakening the N-O bond and lowering ν(N-O).

(47) Ford, P. C.; Lorkovic, I. M. Chem. Rev. 2002, 102, 993–1017.

(48) Walker, F. A.; Simonis, U. Iron Porphyrin Chemistry. In Encyclo-

pedia of Inorganic Chemistry, 2nd ed.; King, R. B., Ed.; John Wiley & Sons Ltd.:

New York, 2005; Vol. IV; pp 2390-2521.

1410 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

Figure 1. EPR spectra of (F₈)Fe(NO)₂(red) and (F₈)FeNO (blue) at the same concentration in acetone, recorded at 77 K.

Table 1. Comparisons of Structural Parameters for Heme Nitrosyl and Related Complexes^a

complex

ν

_NO(solvent), cm^-1

λ_max(nm)

g value

ref

Five-Coordinate

(TPP)Fe(NO)

1670 (KBr), 1678 (CH₂Cl₂)

1685 (KBr)

1688 (KBr)

405 (Soret), 537, 606 (CHCl₃)

g₁= 2.102, g₂= 2.064, g₃= 2.010 39

39

g₁= 2.113, g₂= 2.078, g₃= 2.025 this work

(TPPBr₈)Fe(NO)

(T2,6-Cl₂PP)Fe(NO)

(F₈)Fe(NO)

1684 (Nujol), 1691 (CH₂Cl₂) 400 (Soret), 541 (CH₂Cl₂)

Six-Coordinate

(1-MeIm)(TPP)Fe(NO)

(4-MePip)(TPP)Fe(NO)

(1-MeIm)(F₈)Fe(NO)

(THF)(PPDME)Fe(NO)

(THF)(F₈)Fe(NO)

1625 (KBr)

1653 (KBr)

1624 (KBr)

39

40

g₁= 2.096, g₂= 2.050, g₃= 2.011 41, 42

g₁= 2.100, g₂= 2.063, g₃= 2.011 this work

1669 (THF); 1684 (weak;

due to (F₈)Fe(NO))

410 (Soret), 546 (THF)

(THF)₂(F₈)Fe

(F₈)Fe(NO)₂

(TmTP)Fe(NO)₂

(TPP)Fe(NO)₂

422 (Soret), 544 (THF)

426 (Soret), 538 (acetone)

416 (Soret), 540 (methylcyclohexane)

silent

38

this work

43

1696 (methylcyclohexane)

1695 (CHCl₃)

^aAbbreviations used: TPP = meso-tetraphenylporphyrin; TPPBr₈= octabromo-tetraphenylporphyrin; T2,6-Cl₂PP = 2,6-difluorophenylporphyr-

in; 1-MeIm = 1-methylimidazole; 4-MePip = 4-methylpiperidine; PPDME = protoporphyrin IX dimethyl ester; TmTP = meso-tetra-m-tolyl-por-

phinato dianion.

Figure 2. EPR spectrum of (THF)(F₈)Fe(NO) recorded at 77 K in THF with g₁= 2.100, g₂= 2.063, and g₃= 2.011. It is essentially identical to the

spectrum of (THF)(PPDME)Fe(NO), reported by Yoshimura.⁴²

the radical species (F₈)Fe(NO) (S = 1/2) by the addition

of another radical, •NO. As discussed below, NMR

spectroscopic data are also consistent with the diamag-

netism of (F₈)Fe(NO)₂.

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1411

Figure 3. UV-vis spectrum of (F₈)Fe(NO)₂(blue, λ_max= 426 (Soret), 538 nm) generated from(F₈)Fe^II(red, λ_max= 421 (Soret), 553 nm) inacetone at-80

ꢀC. Uponwarmingthe solution (in a closedcuvette glassware apparatus), the mononitrosylspecies (F₈)Fe(NO) reforms (green, λ_max= 399 (Soret), 540 nm).

Table 2. ¹H NMR Spectroscopic Resonance Comparison: Heme-Fe Mono- and

Dinitrosyl Complexes (CD₂Cl₂at RT)

þ NO

warm up

ðF₈ÞFeðNOÞ

ðF₈ÞFeðNOÞ₂

ð2Þ

pyrrole-H

(ppm)

meta-H

(ppm)

para-H

(ppm)

In fact, the binding of •NO_(g)to (F₈)Fe(NO) is rever-

sible, as can be monitored by EPR and UV-vis spectro-

scopies. Warming of an EPR sample of (F₈)Fe(NO)₂,

removing gases in vacuo, and refreezing and now record-

ing an EPR spectrum leads to regeneration of most of the

triplet EPR spectrum due to (F₈)Fe(NO). A UV-vis

spectrum of (F₈)Fe(NO) shows absorptions at 399

(Soret) and 541 nm in acetone, which change when excess

NO_(g)is bubbled through the cold solution, forming

(F₈)Fe(NO)₂(Figure 3). There is no obvious color change

of the reaction solution in either solvent. When

(F₈)Fe(NO)₂is warmed to RT, whether excess •NO_(g)is

present or not, (F₈)Fe(NO) is reformed (eq 2).

To further confirm the formulation of the dinitrosyl

complex (F₈)Fe(NO)₂, we added 1 equiv of the reduced

compound (F₈)Fe^IIto a low-temperature solution of

(F₈)Fe(NO)₂from which excess •NO_(g)had been removed

via vacuum/purge cycles. There is some immediate trans-

fer to give a mixture of mono- and dinitrosyl complexes,

based on UV-vis spectroscopy (Figure S4, Supporting

Information). However, warming to RT results in the full

formation of 2 equiv of (F₈)Fe(NO); thus, the overall

reaction proceeded as follows: (F₈)Fe(NO)₂þ (F₈)-

Fe^IIf 2 (F₈)Fe(NO). Not only does this experiment

demonstrate that our low-temperature species is a dini-

trosyl complex, that is, possessing two NO molecules per

iron, but that the second NO molecule binds much more

weakly than the first and can be extracted by another

heme molecule.

heme-Fe complex

ref

(TPP)Fe(NO)

(TMP)Fe(NO)

(F₈)Fe(NO)

5.95

5.79

6.00

8.25

7.45

40

43

8.42/8.22

8.17

8.2

7.39

8.18

7.65

6.7

7.19

7.67

7.86

7.85

(OEP)Fe(NO)^a

(OEP)Fe(NO)₂

a

8.94

6.05

9.12/8.54

9.13/8.93

(F₈)Fe(NO)

(F₈)Fe(NO)^b

(F₈)Fe(NO)₂

this work

7.45

b

^aSpectra recorded in toluene-d₇. ^bSpectra recorded at -80 ꢀC.

splitting to one sharp peak at 9.12 ppm and one broad

peak at 8.54 ppm, with integration found to be in a 1:7

ratio. With further cooling, the integration ratio of sharp

peak versus broad peak increases to 2:6 (at -40 ꢀC), 3:5 (at

-60 ꢀC), and 4:4 (at -80 ꢀC). This trend is in accordance

with a slowing of the NO rotation or even a “sitting”

oxygen atom behavior. Especially at -80 ꢀC, four pyrrole

protons were expected to be in the exact same magnetic

environment, and another four pyrrole protons stayed in

a very similar environment, due to two equally occupied

NO orientations at low temperatures, as Scheidt and co-

workers⁴⁹suggested in the crystalline phase studies of

(TPP)Fe(NO). The assignments of these peaks as pyrrole

2

resonances were also confirmed by H NMR spectros-

copy employing a deuteriated heme complex, (F₈-d₈)Fe-

(NO) (see Figure S6, Supporting Information).

The H NMR spectrum of what was thought to be

1

(F₈)Fe(NO)₂, as recorded at -80 ꢀC, presents what

appears to be a symmetrically split signal for the pyrrole

protons (Figure 4). However, the peak at 9.13 ppm over-

laps exactly with that of the mononitrosyl species

(F₈)Fe(NO), indicating that a mixture is present and that

the 8.93 ppm signal can be assigned to the dinitrosyl

species. With warming to -60 ꢀC and -40 ꢀC, ¹H NMR

spectra (Figure S7, Supporting Information) indicate that

a mixture of (F₈)Fe(NO)₂and (F₈)Fe(NO) is present, but

with less dinitrosyl complex present. The 8.93 ppm signal

diminishes and shifts/broadens. At RT, the mononitrosyl

NMR Spectroscopy for the Heme-Nitrosyl Complexes.

At RT, a ¹H NMR spectrum of (F₈)Fe(NO) gives a broad

pyrrole proton signal at 6.05 ppm (Table 2), consistent

with that found for other heme mononitrosyl compounds

(Table 2).⁴⁰Rather interesting temperature-depen-

dent behavior occurs, as is now described along with

tentative interpretations. As shown in Figure 4 and

1

Figure S5 (Supporting Information), H NMR spectra

of (F₈)Fe(NO) were recorded at þ20 ꢀC, -20 ꢀC, -40 ꢀC,

-60 ꢀC, and -80 ꢀC. At 20 ꢀC, the pyrrole resonance is a

broad peak, which may be due to the fast rotation of NO

about the Fe-NO bond. When cooling to ∼-20 ꢀC, a

striking downfield shift occurs accompanying pyrrole

(49) Silvernail, N. J.; Olmstead, M. M.; Noll, B. C.; Scheidt, W. R. Inorg.

Chem. 2009, 48, 971–977.

1412 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

Figure 5. Two possible conformations (cis, trans) of the present

(F₈)Fe(NO)₂complex. Both have been suggested, but DFT calculations

in 2003 from the groups of both Ford⁵⁸and Ghosh^59,60support the view

that the cis configuration would be the preferred structure in a porphyr-

inate-iron environment.

(TmTP = meso-tetra-m-tolyl-porphinato dianion) and

Fe(TPP)(NO)₂, using cryogenic conditions to stabilize

the binding of a second NO; the adducts were charac-

terized by UV-vis, IR, and NMR spectroscopies. Fe-

(TmTP)(NO) displays ν_(N-O)= 1683 cm^-1; Fe(TmTP)-

(NO)₂also possesses a single N-O stretch, ν_(N-O)

=

1696 cm^-1

In more biological situations, a trans-dinitrosyl species

.

had been suggested to occur during (NO)Fe^IIIhemoglo-

bin autoreduction chemistry,⁵³while to explain •NO_(g)

-

dependent behavior during s-guanylate cyclase (sGC)

activity, Ballou and co-workers⁵⁴at one point raised the

possibility of such an entity forming. Since then, biophy-

sical^55,56and theoretical studies⁵⁷on cytochrome

c⁰(cyt. c⁰), bacterial class II cytochromes perhaps involved

in •NO_(g)detoxification, led to suggestions that its heme

may bind two NO’s. As there are similarities between cyt.

c⁰and sGC (e.g., they both bind NO but not O₂),⁵⁷the cyt.

c⁰researchers have supported the view that a Fe(NO)₂

species may form as a transient intermediate in sGC.

In fact, two computational studies^58-60carried out

have led to a proposed heme dinitrosyl complex structure,

both suggesting a lowest-energy “trans-syn” conforma-

tion,^59,60what we refer to here as the “cis”⁵⁸conforma-

tion, Figure 5. Here, the nitrosyl ligands reside on

opposite sides of the heme and the NO groups are bent

over in the same direction. While Ford and Lorkovic⁴³

only observed a single pyrrole resonance for Fe(TmTP)-

(NO)₂, it is notable that here we observe two types of

protons for (F₈)Fe(NO)₂at -80 ꢀC, Figure 4. However,

these data still cannot differentiate between two possible

structures, the “cis” versus the “trans” configuration

(Figure 5).

Figure 4. ¹H NMR spectra recorded in CD₂Cl₂. Top: (F₈)Fe(NO), RT.

Middle: (F₈)Fe(NO) at -80 ꢀC. Bottom: (F₈)Fe(NO)₂at -80 ꢀC. The

peak at 5.32 ppm is CD₂Cl₂.

complex fully reforms.⁵⁰These results indicate that,

under the conditions of the H NMR experiment, with

1

the concentrations of heme and the amount of NO_(g)that

could be added to the sample tube, (F₈)Fe(NO)₂is not

fully formed. This is unlike what the situation appears to

be in the separate UV-vis and EPR spectroscopy experi-

ments (vide supra), where full formation is indicated.

A number of heme dinitrosyl complexes have been

previously described, and there has been increased inter-

est in such species. In 1974, Wayland and Olson⁵¹sug-

gested that a dinitrosyl complex forms from the reversible

coordination of •NO_(g)to (TPP)Fe(NO) in toluene. The

compound was suggested to be a (TPP)Fe^II(NO^-)(NO^þ)

species, that is, with a linear Fe^II-NO^þunit and a bent

Fe^II-NO^-unit. In an electrochemical study in 1983,

Kadish and Lancon⁵²suggested that the formation of

To summarize, we have observed here the reversible

formation of (F₈)Fe(NO)₂from a •NO_(g)reaction with

(F₈)Fe(NO), as deduced via UV-vis, EPR, and NMR

(OEP)Fe(NO)₂(OEP

= octaethylporphyrin) and

(53) Addison, A. W.; Stephanos, J. J. Biochemistry 1986, 25, 4104–4113.

(54) Zhao, Y.; Brandish, P. E.; Ballou, D. P.; Marletta, M. A. Proc. Natl.

Acad. Sci. 1999, 96, 14753–14758.

(55) Lawson, D. M.; Stevenson, C. E. M.; Andrew, C. R.; Eady, R. R.

Embo. J. 2000, 19, 5661–5671.

(56) Pixton, D. A.; Petersen, C. A.; Franke, A.; van Eldik, R.; Garton,

E. M.; Andrew, C. R. J. Am. Chem. Soc. 2009, 131, 4846–4853.

(57) Marti, M. A.; Capece, L.; Crespo, A.; Doctorovich, F.; Estrin, D. A.

J. Am. Chem. Soc. 2005, 127, 7721–7728.

(TPP)Fe(NO)₂occurs under •NO_(g)pressure and that

these could be electrochemically oxidized to cationic

species. Ford and Lorkovic⁴³pointed out that the optical

spectrum for that TPP complex more resembled that of a

nitrosyl/nitrito complex. In 2000, Ford and Lorkovic⁴³

described the dinitrosyl compounds Fe(TmTP)(NO)₂

(58) Patterson, J. C.; Lorkovic, I. M.; Ford, P. C. Inorg. Chem. 2003, 42,

4902–4908.

(59) Conradie, J.; Wondimagegn, T.; Ghosh, A. J. Am. Chem. Soc. 2003,

125, 4968–4969.

(60) Ghosh, A. Acc. Chem. Res. 2005, 38, 943–954.

(50) ¹H NMR spectra of (F₈)Fe(NO)₂and (F₈)Fe(NO) in acetone-d₆are

also given in the Supporting Information.

(51) Wayland, B. B.; Olson, L. W. J. Am. Chem. Soc. 1974, 96, 6037–6041.

(52) Lancon, D.; Kadish, K. M. J. Am. Chem. Soc. 1983, 105, 5610–5617.

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1413

Table 3. UV-Vis Comparison of Relevant (F₈)-Fe Complexes

complex

THF (λ_max, nm)

acetone (λ_max, nm)

(F₈)Fe(NO)

(F₈)Fe(NO)₂

410 (Soret), 546

410 (Soret), 540

394 (Soret), 510

410 (Soret), 546

399 (Soret), 541

426 (Soret), 538

404 (Soret), 510, 568

399 (Soret), 541

[(F₈)Fe^III]SbF₆

products of {(F₈)Fe(NO)₂þ (tmpa)Cu(MeCN)]^þþ 2 H^þ}

products of {(F₈)Fe(NO)₂þ (tmpa)Cu(MeCN)]^þ}

spectroscopic studies.⁶¹Future investigations will be

directed at understanding more about the electronic

structure of (F₈)Fe(NO)₂or analogs (such as studying

solution upon addition of the copper(I) complex and acid

(see the Experimental Section). A UV-vis spectrum of

the product mixture has λ_max= 394 (Soret) and 510 nm

(in THF, Table 3), which corresponds to what should be a

heme-Fe^IIIcomplex [(F₈)Fe^III]^þwith a B(C₆F₅)₄^-coun-

teranion (see the Experimental Section). In fact, these

UV-vis properties perfectly match those of [(F₈)-

Fe^III]SbF₆, which was independently synthesized

(Table 3). To further determine the identity of the pro-

ducts of the reaction (eq 3), an EPR spectrum was

recorded following precipitation of the solids from the

reaction mixture in acetone/CH₃CN and redissolution in

acetone (Figure 6A). It also confirms the presence of the

heme-Fe^IIIspecies (i.e., [(F₈)Fe^III]^þ) along with a Cu^II

complex with trigonal bipyramidal (TBP) coordination

geometry, possessing well-known (for TBP Cu^II) “re-

verse” axial EPR spectral properties (g_^> g ).^62-64In

fact, this perfectly matches the known spectroscopic

behavior of [(tmpa)Cu^II(MeCN)]^2þ(see the Experimental

Section and Figure 6B). To confirm that the heme and

copper products were formed in equal amounts, as per eq

3 and Scheme 3A, we generated and recorded an EPR

spectrum of a made-up 1:1 (molar equiv) mixture of

€

Mossbauer spectroscopic properties), the detailed nature

of the equilibrium between it and its mononitrosyl pre-

cursor, and the reactivity of (F₈)Fe(NO)₂with nucleo-

philic or electrophilic substrates. As mentioned above, the

dinitrosyl complex has been very useful for us to carry out

stoichiometric reactions where only two •NO_(g)molecules

are present for a heme plus copper complex reaction, as

described below.

Reaction of (F₈)Fe(NO)₂with [(tmpa)Cu^I(MeCN)]^þ

Plus Acid; •NO Reductive Coupling. With the presence

of 2 equiv of acid in the form of H(Et₂O)₂[B(C₆F₅)₄]

(HBArF), the reductive coupling of nitrogen monoxide

was efficiently facilitated by this heme and copper com-

plexes mixture of (F₈)Fe(NO)₂and [(tmpa)Cu^I(MeCN)]^þ

(Scheme 3A), in the same stoichiometry as is known for

CcOs:

ðF₈ÞFe^IIþ ½ðtmpaÞCu^IðMeCNÞꢁ^þþ 2NO

ðgÞ

þ 2H^þf ½ðF₈ÞFe^IIIꢁ^þþ ½ðtmpaÞCu^IIðMeCNÞꢁ

2þ

[(F₈)Fe^III]SbF₆

and

[(tmpa)Cu^II(MeCN)](ClO₄)₂,

þ N₂O þ H₂O

ð3Þ

ðgÞ

Figure 6B. This beautifully matches the spectrum re-

corded for the product mixture in the NO reductive

coupling reaction (Figure 6A), (F₈)Fe^III(g = 6.10) and

Cu^II(g_^= 2.21, g = 2.01, A_^= 100 G, A = 68 G).

To check on other possible products in the reaction

mixture, we used the solid material obtained by adding

pentane to the acetone/MeCN reaction mixture. An IR

spectrum (Nujol mull)⁶⁵showed that there is no nitro-

syl-heme species present, further corroborating the nat-

ure of the reaction; both NO molecules initially present as

the (F₈)Fe(NO)₂complex are consumed, and N₂O_(g)is

produced (vide supra). To check for the possibility that

nitrite might have been generated and might have ac-

counted for some product of •NO reactivity, we carried

out ion chromatography analysis on an aqueous extract

of the reaction mixture (see the Experimental Section).

However, no trace of nitrite could be detected. A room-

temperature ¹H NMR spectrum of the products solution

in CD₃CN (Figure 10) showed it was a mixture of what

appeared to be a high-spin heme-Fe^IIIcomplex and also

possessing paramagnetically shifted signals ascribed

to the TMPA ligand on [(tmpa)Cu^II(MeCN)](ClO₄)₂

Gas chromatography analysis (see the Supporting In-

formation, Figure S11) of the head space of the product

mixture revealed a N₂O_(g)yield of 84%, based on eq 3 and

corresponding to the NOR stoichiometry of 2NO þ 2H^þ

þ 2e^-f N₂O þ H₂O. This compares to the 80% yield

of gaseous nitrous oxide obtained in the system with a

binuclear heme/Cu complex of the ⁶L ligand (Scheme 2);¹

both the binuclear complex and present component

(heme þ copper complex) system efficiently effect the

NOR chemistry.

In order to rule out the possibility that only

(F₈)Fe(NO)₂itself mediates the •NO reductive coupling

observed, its reaction with 2 equiv of acid (HBArF) in a

cold acetone solution was investigated. No UV-vis

change was observed at low temperatures, even following

warming from -80 ꢀC. The iron(II) nitrosyl complex

(F₈)Fe(NO) was produced as determined by UV-vis,

IR, and EPR spectroscopic analysis; •NO_(g)was released

as detected by GC analysis. This control experiment

indicates that only (F₈)Fe(NO)₂and acid cannot facilitate

NO reductive coupling; that is, the copper complex

[(tmpa)Cu^I(MeCN)]^þhas a critical role in the NOR

chemistry described here.

(62) Maiti, D.; Lee, D.-H.; Narducci Sarjeant, A. A.; Pau, M. Y. M.;

Solomon, E. I.; Gaoutchenova, K.; Sundermeyer, J.; Karlin, K. D. J. Am.

Chem. Soc. 2008, 130, 6700–6701.

(63) Lucchese, B.; Humphreys, K. J.; Lee, D.-H.; Incarvito, C. D.;

Sommer, R. D.; Rheingold, A. L.; Karlin, K. D. Inorg. Chem. 2004, 43,

5987–5998.

Identity of Products Formed. Different from the start-

ing material employed, the orange-colored dinitrosyl

complex (F₈)Fe(NO)₂forms a homogeneous brown

(64) Karlin, K. D.; Hayes, J. C.; Shi, J.; Hutchinson, J. P.; Zubieta, J.

Inorg. Chem. 1982, 21, 4106–4108.

(65) Data not shown.

(61) We have thus far been unable to obtain low-temperature infrared

spectroscopic data for this complex.

1414 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

oxidases, since our research group has synthesized and

published on a number of heme-Fe^III-O-Cu^IIcom-

plexes, including ones which are structurally character-

ized.^29,70,71So, perhaps the heme-iron and copper ions

would facilitate •NO_(g)coupling chemistry and also be

Lewis acidic enough to force this reaction without pro-

tons, as

heme-Fe^IIþ Cu^I_Bþ 2•NO

ðgÞ

f heme-Fe^III-O-Cu^II_Bþ N₂O

ðgÞ

However, this is not the case, and as described above, acid

is required to effect •NO_(g)coupling in the (F₈)Fe(NO)₂þ

[(tmpa)Cu^I(MeCN)]^þsystem. In fact, when [(tmpa)-

Cu^I(MeCN)]^þis added to (F₈)Fe(NO)₂without acid

present, the products obtained are a mixture of heme-

nitrosyl plus Cu^II-nitrite and unreacted Cu^Icomplexes,

eq 4 (and Scheme 3B).

ðF₈ÞFeðNOÞ₂þ ½ðtmpaÞCu^IðMeCNÞꢁ^þf ðF₈ÞFeðNOÞ

þ

-

þ 1=3½ðtmpaÞCu^IIðNO₂Þꢁ

þ 2=3½ðtmpaÞCu^IðsolventÞꢁ^þþ 1=3N₂O

ð4Þ

ðgÞ

Figure 6. EPR spectra (acetone; 77 K) of (A) the product mixture of the

reaction (F₈)Fe(NO)₂þ [(tmpa)Cu^I(MeCN)]^þþ 2 H^þ(blue) show-

ing the typical heme-Fe^IIIcomplex and [(tmpa)Cu^II(solvent)]^2þ; (B) a

madeup 1:1 mixture of [(F₈)Fe^III]SbF₆and [(tmpa)Cu^II(MeCN)](ClO₄)₂

(brown); (C) the product mixture obtained from the reaction of

(F₈)Fe(NO)₂þ [(tmpa)Cu^I(MeCN)]^þin the absence of acid (red),

(F₈)Fe(NO) þ 1/3[(tmpa)Cu^II(NO₂^-)]^þþ unreacted 2/3[(tmpa)Cu^I-

(solvent)]^þ. See Figure S8 (Supporting Information) for an expanded

view of this spectrum. See the text for further explanation.

A UV-vis spectrum of a reaction mixture reveals

bands associated with the presence of (F₈)Fe(NO),

only λ_max(THF) = 410 (Soret) and 546 nm and λ_max

(acetone) = 399 (Soret) and 541 nm, and IR spectroscopy

gives ν_NO= 1684 cm^{-1 65}

An EPR spectrum of the

.

reaction product solution (Figure 6C) indicates that it is

a mixture of typical (porphyrinate)Fe^II-NO and Cu^II

species,⁷²consistent with our proposed course of reaction

(eq 4). Thus, without added acid, one of the two nitrogen

monoxide molecules (per heme/Cu stoichiometric

mixture) remains within a metal complex product, that

is, in the form of (F₈)Fe(NO). Our expectation then was

that any remaining •NO released may have undergone a

copper complex mediated disproportionation reaction,

giving nitrite and N₂O_(g)according to well-established

copper(I)/•NO_(g)chemistry:^4,45

(as determined from the previously determined NMR

properties of this complex).⁶⁶A more detailed description

of the NMR spectroscopic and spin-state properties of

this heme complex, [(F₈)Fe^III]SbF₆, will be discussed

below.

Reaction of (F₈)Fe(NO)₂with [(tmpa)Cu^I(MeCN)]^þin

the Absence of Acid. Of course, the presence of protons is

required in NORs to give turnover, see eq 1. In fact, we

originally hypothesized that we may not need protons to

effect •NO reductive coupling. In NORs, it is thought that

a μ-oxo heme/nonheme diiron(III) complex forms follow-

ing reductive coupling, as this has been detected by

several groups.^4,11,67-69Perhaps the chemistry could

proceed as follows:

þ

-

3•NO þ ½ðLÞCu^Iꢁ f N₂O þ ½ðLÞCu^IIðNO₂Þꢁ

ð5Þ

In fact, this seems to be the case. GC analysis reveals

that N₂O_(g)is produced in 88% yield on the basis of eq 5.

This is far less than should be or is produced by our NOR

chemistry with acid present, Scheme 3A and eq 3. In

further support of the chemistry outlined here, ion chro-

matography analysis of an aqueous solution extract of the

reaction mixture (see the Experimental Section) indicates

that nitrite (NO₂^-) is present in the product mixture with

a yield of 95%, according to eq 5. In summary, without

added acid, one of the two nitrogen monoxide molecules

heme-Fe^IIþ Fe_Bþ 2•NO f heme-Fe -O-Fe^III

III

II

B

þ N₂O

ðgÞ

Thus, we hypothesized that proton equivalents might not be

necessary for the same chemistry to occur in heme-copper

(70) Ju, T. D.; Ghiladi, R. A.; Lee, D.-H.; van Strijdonck, G. P. F.;

Woods, A. S.; Cotter, R. J.; Young, J. V. G.; Karlin, K. D. Inorg. Chem.

1999, 38, 2244–2245.

(66) Nanthakumar, A.; Fox, S.; Murthy, N. N.; Karlin, K. D. J. Am.

Chem. Soc. 1997, 119, 3898–3906.

(67) Pinakoulaki, E.; Gemeinhardt, S.; Saraste, M.; Varotsis, C. J. Biol.

Chem. 2002, 277, 23407–23413.

(71) Kim, E.; Helton, M. E.; Wasser, I. M.; Karlin, K. D.; Lu, S.; Huang,

€

H.-w.; Moenne-Loccoz, P.; Incarvito, C. D.; Rheingold, A. L.; Honecker,

(68) Moenne-Loccoz, P.; Richter, O.-M. H.; Huang, H.-w.; Wasser, I. M.;

€

Ghiladi, R. A.; Karlin, K. D.; de Vries, S. J. Am. Chem. Soc. 2000, 122, 9344–

9345.

M.; Kaderli, S.; Zuberbuhler, A. D. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,

3623–3628.

(69) Field, S. J.; Prior, L.; Roldan, M. D.; Cheesman, M. R.; Thomson,

A. J.; Spiro, S.; Butt, J. N.; Watmough, N. J.; Richardson, D. J. J. Biol.

Chem. 2002, 277, 20146–20150.

(72) The amount of Cu(II) complex present should be at a level of 1/3 of

the amount of heme-nitrosyl complex present, see eq 4 thus, the g = 2.0-2.2

region of Figure 6C is dominated by the heme-nitrosyl complex signal

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1415

remains in the product, and the other •NO_(g)molecule

released is disproportionated to nitrite in the form of a

nitrito complex and N₂O_(g). Further evidence comes from

control experiments, where we separately show that

[(tmpa)Cu^I]^þis a complex which does facilitate nitrogen

monoxide disproportionation, see below.

Reaction of [(tmpa)Cu^I(MeCN)]^þwith •NO_(g); Nitrogen

Monoxide Disproportionation^4,45. When excess •NO_(g)is

exposed to [(tmpa)Cu^I(MeCN)]^þ, the related nitrito com-

plex [(tmpa)Cu^II(NO₂)]^þis produced in excellent yield, as

determined by elemental analysis and UV-vis and EPR

spectroscopies (Figures S2 and S3, Supporting In-

formation) and ion chromatography, which was carried

out to further confirm/identify the nitrite anion (see the

Experimental Section). Nitrous oxide gas was also iden-

tified qualitatively (by GC); quantitative determination

was made difficult by the conditions of excess •NO(g)

required to obtain high yields of [(tmpa)Cu^II(NO₂)]^þ. To

summarize, [(tmpa)Cu^I(MeCN)]^þis capable of •NO_(g)

disproportionation chemistry and is likely the source of

N₂O_(g)when [(tmpa)Cu^I(MeCN)]^þreacts with •NO_(g), by

itself (as described here), or in the reaction also with

(F₈)Fe(NO)₂present but without added acid (vide supra).

Structure, Spectroscopy, and Spin-State Properties of

[(F₈)Fe^III](SbF₆). As described above, [(F₈)Fe^III]^þ(as

the B(C₆F₅)₄salt) was formed in the NOR model

system reacting (F₈)Fe(NO)₂with [(tmpa)Cu^I(CH₃-

Figure 7. ORTEP diagram showing the cationic portion of of

[(F₈)Fe^III(THF)₂]SbF₆. Also see Table 4 for structural parameters.

known to be spin-admixed complexes,³⁵also see

Table 4. Walker and co-workers⁷⁴have in fact previously

studied [(F₈)Fe^III]ClO₄; in CH₂Cl₂, this was shown to be

an S = 3/2 and S = 5/2 spin admixture. The ground state

was largely high-spin with the S = 3/2 state as the first

excited state, on the basis of EPR (4.2 K) and NMR

(CD₂Cl₂) spectroscopic properties. Here, we compare

these properties with our own [(F₈)Fe^III]SbF₆in CH₂Cl₂.

As we were able to obtain crystals and an X-ray structure

of [(F₈)Fe^III(THF)₂]SbF₆(vide infra), we also more care-

fully examined this six-coordinated species’ THF solution

EPR (77 K) and variable-temperature NMR spectro-

scopic properties. As described below, we also find both

[(F₈)Fe^III]SbF₆and [(F₈)Fe^III(THF)₂]SbF₆complexes to

be an S = 3/2 and S = 5/2 spin admixture.

CN)]^þB(C₆F₅)₄in the presence of acid. And to help

-

confirm its identity, we separately synthesized [(F₈)-

Fe^III]SbF₆via a metathesis reaction of (F₈)Fe^IIICl with

AgSbF₆(see the Experimental Section and Figure S9,

Supporting Information), and we compared UV-vis and

EPR properties with those obtained in our NOR model

reaction. However, a closer examination of the EPR and

NMR properties of [(F₈)Fe^III]SbF₆indicates that the

complex exists as a spin-state mixture of S = 3/2 and

S = 5/2, with the ground state being mainly a high-spin

state (S = 5/2). The following paragraphs detail this

situation.

Recrystallization of isolated complex [(F₈)Fe^III]SbF₆

out of THF/hexane provides a bis-THF complex,

[(F₈)Fe^III(THF)₂]SbF₆(see the Experimental Section

and Supporting Information); the structure is depicted

in Figure 7. The average Fe-N_porphyrinatebond distance is

˚

2.027 (3) A, clearly between those of known low-spin

˚

(avg. = ∼1.99 A) and high-spin six-coordinate deriva-

˚

tives (avg. > 2.04 A). Also see Table 4 for these and other

relevant comparisons. The value is also much shorter

There is extensive literature concerning spin states

which can occur with iron(III) porphyrinate com-

plexes.^48,73High-spin, intermediate-spin, or low-spin

states which occur are dramatically influenced by the

external ligand(s) bound to the heme. A variety of phy-

sical techniques can be applied to characterize and differ-

entiate spin-state possibilities for heme-Fe^IIIcomplexes,

such as X-ray crystallography along with EPR and NMR

spectroscopies. One of the principal structural features

is the M-N_porphyrinatebond length, which increases as

one goes from lower to higher spin states, that is, S = 1/2

< S = 3/2 < S = 5/2. Second, g values observed in EPR

spectra become enlarged when the spin states are

higher. Third, ¹H NMR spectra exhibit notable downfield

shifts of the pyrrole protons when the spin states

change from low to intermediate to high spin. Certain

heme-Fe^IIIcomplexes are known to possess an admix-

ture (but not thermal equilibrium) of S = 3/2 and S =

5/2 species, and these exhibit a range of physical pro-

perties.^48,73

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(82) Scheidt, W. R.; Cohen, I. A.; Kastner, M. E. Biochemistry 1979, 18,

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(84) Higgins, T. B.; Safo, M. K.; Scheidt, W. R. Inorg. Chim. Acta 1990,

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Scheidt, W. R. Inorg. Chem. 1994, 33, 1319–1324.

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Eaton, G. R. Inorg. Chem. 1992, 31, 5450–5465.

In fact, a good number of [(porphyrinate)Fe^III]^þcom-

plexes, such as five-coordinated [(TPP)Fe^III]ClO₄, are

(73) Scheidt, W. R. Porphyrin Hand. 2000, 3, 49–112.

1416 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

Table 4. Selected Fe-N Bond Distances, g Values, and ¹H NMR of heme-Fe^IIIComplexes^a

heme-Fe^IIIcomplex

Fe-N (A)

g_^value

pyrrole-H (δ, ppm)

spin state

reference

˚

Five-Coordinate

(OEP)FeCl

(TPP)FeCl

(F₈)FeCl

[(OEP)Fe](ClO₄)

[(TPP)Fe](ClO₄)

[(F₈)Fe](ClO₄)

[(F₈)Fe](SbF₆)

2.063

5.76 (toluene)

5.66 (toluene)

6.16 (CH₂Cl₂)

5.83 (CH₂Cl₂)

4.72 (CH₂Cl₂), 5.84 (2-metTHF)

5.72 (CH₂Cl₂)

5.84 (CH₂Cl₂)

80.3 (CDCl₃)

80.8 (toluene-d₈)

81.0 (CDCl₃)

h.s.^b

h.s.

i.s.^c

h.s. þ i.s.

73, 75, 76

75, 77, 78

29, 30

79,80

35, 74

2.040 (9) 2.070 (9)

2.089(4)

1.994(10)

2.001(5)

-10

∼15

74

this work

27.2, 11.6

Six-Coordinate

[(OEP)Fe(DMSO)₂]^þ

[(TPP)Fe(H₂O)₂]^þ

2.035(9)

h.s.

81

2.045(8) 2.029(5)

2.004(2)

1.982(3)

1.999(2) 1.978(12)

2.00(1)

2.027(3)

∼6 (solid)

h.s.

82, 83

81

84

85, 86

87

this work

[(OEP)Fe(1-MeIm)₂]^þ

[(TPP)Fe(1-MeIm)₂]^þ

[(OEP)Fe(THF)₂]^þ

[(TPP)Fe(THF)₂]^þe

[(F₈)Fe(THF)₂](SbF₆)

l.s.^d

l.s.

g_z= 2.890, g_y= 2.291, g_x= 1.554

4.68 (CH₂Cl₂)

i.s.

h.s þ i.s.

6.12 (THF)

54.7, 14.8

^aOEP = octaethylporphine; TPP = meso-tetraphenylporphyrin; 2-metTHF = 2-methytetrahydrofuran; 1-MeIm = 1-methylimidazole; DMSO =

dimethyl sulfoxide. ^bh.s. = high spin, S = 5/2. ^ci.s. = intermediate spin, S = 3/2. ^dl.s. = low spin, S = 1/2. ^eDerived from a dinuclear heme-Fe^III-Cu^II

complex, [(TPP)Fe(THF)₂]-{[(TPP)Fe(THF)][Cu(MNT)₂]} 2THF (MNT = cis-1,2-dicyanoethylenedithiolate).

3

than that of the related five-coordinated high-spin com-

state for both [(F₈)Fe^III]SbF₆and [(F₈)Fe^III(THF)₂]SbF₆,

while the latter is almost completely at this state.

30

plex (F₈)Fe^IIICl (Fe-N_porphyrinate= 2.089(4) A), the

˚

material used to synthesize [(F₈)Fe^III(THF)₂]SbF₆. In

Further, the ¹H NMR spectrum of [(F₈)Fe^III]SbF₆and

the position of the pyrrole proton resonances indicates

the complex to be in a mixed-spin state at RT in CD₂Cl₂,

Figure S10 (Supporting Information). Two pyrrole reso-

nances are observed at δ_pyrrole= 27.2 and 11.6 ppm, both

typical for intermediate-spin complexes. These shift

downfield when the temperature decreases but still lie in

the intermediate-spin state range. [(F₈)Fe^III]ClO₄exhibits

˚

addition, the axial Fe-O_THFbond distance, 2.131 A, is

significantly longer than those observed for low-spin state

complexes, but very similar to those of two other bis-THF

complexes, [(TPP)Fe(THF)₂]^þ(Fe-O = 2.16 A) and

˚

[(OEP)Fe(THF)₂]^þ(Fe-O = 2.187 A; Table 4). As

˚

stated, these compounds are known to possess intermedi-

ate-spin states. The possibility that [(F₈)Fe^III]SbF₆is not

a quantum-admixed spin system, but rather possesses an

intermediate-spin system, is ruled out by EPR and NMR

spectroscopic information, as described below.

δ

_pyrrole=∼15 ppm at RT).⁷⁴In THF-d₈, signals for both high

spin (δ_pyrrole= 54.7 at RT) and intermediate spin (δ_pyrrole

=

14.8 at RT) are observed at all temperatures (Figure 9).

However, a greater fraction of high-spin [(F₈)Fe^III-

(THF)₂]SbF₆species is present at reduced temperatures,

When [(F₈)Fe^III]SbF₆, lacking any THF, is dissolved in

CH₂Cl₂and the EPR spectrum recorded at 77 K, a g =

5.84 feature is observed (Figure 8). This lies in the range

expected for admixed-spin state complexes (g = 4.2-6)

and is not typical for high- (g = ∼ 6.2) or low-spin

complexes (g = ∼3.5 or <3.4).⁷³Our g = 5.84 signal

also well matches that observed for [(F₈)Fe^III]ClO₄(g =

5.72, 4.2 K).⁷⁴In addition, this EPR spectrum of

[(F₈)Fe^III]SbF₆in frozen CH₂Cl₂is much broader than

that of the typical high-spin complex (F₈)Fe^IIICl

(Figure 8). The broadening has been suggested to be

due to a quantum admixed feature.⁸⁸In the strongly

coordinating solvent THF, the 77 K EPR spectrum of

[(F₈)Fe^III(THF)₂]SbF₆indicates that the complex is

mostly or all high-spin, based on the observed g = 6.12

(Figure 8). In other words, and according to the criterion

δ_pyrrole(high-spin) = 54.7 ppm at RT, shifting to 107 ppm

at -80 ꢀC, which is in the high-spin chemical shift region.

To summarize, the ¹H NMR spectra are consistent with

EPR spectroscopic and X-ray structural parameters; that

is, the ground states of both [(F₈)Fe^III]SbF₆and

[(F₈)Fe^III(THF)₂]SbF₆are high-spin (S = 5/2), while

[(F₈)Fe^III]SbF₆has a larger intermediate-spin fraction

(S = 3/2) with increasing temperature. As mentioned

1

earlier, a H NMR spectrum of the product mixture of

{(F₈)Fe(NO)₂þ [(tmpa)Cu^I(MeCN)]^þþ 2 H^þ} in

CD₃CN (Figure 10) might suggest that a high-spin

[heme-Fe^III]B(C₆F₅)₄(vide supra) product is formed,

which seems inconsistent with the data just described

for the synthetically derived [(F₈)Fe^III]SbF₆(Figure S10,

Supporting Information) or [(F₈)Fe^III(THF)₂]SbF₆species.

While considering the controlling role of exogenous ligands

on the spin states of six-coordinate heme-Fe^IIIcomplexes,

it is likely that either a water molecule (produced from the

NOR type reaction) or acetonitrile or acetone (from the

reaction solvents) binds to (F₈)Fe^III, and this results in it

being mostly in a high-spin state.

put forth by Walker and co-workers⁸⁹along with Mal-

2

tempo and Moss,⁹⁰g_^= 6(a_5/2)²þ 4(b_3/2)²{where (a_5/2

)

and (b_3/2)²are the coefficients of the high-spin and inter-

mediate-spin states, respectively}, and thus the EPR data

suggest that the high-spin state (S =5/2) is the ground

(88) Gismelseed, A.; Bominaar, E. L.; Bill, E.; Trautwein, A. X.; Winkler,

H.; Nasri, H.; Doppelt, P.; Mandon, D.; Fischer, J.; Weiss, R. Inorg. Chem.

1990, 29, 2741–2749.

Summary/Conclusions: Implications for NORs

(89) Yatsunyk, L. A.; Shokhirev, N. V.; Walker, F. A. Inorg. Chem. 2005,

44, 2848–2866.

(90) Maltempo, M. M.; Moss, T. H. Q. Rev. Biophys. 1976, 9, 181–215.

In this report, we have described heme/Cu complex com-

ponents (in a 1:1 mixture) facilitating •NO_(g)reduction to

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1417

Figure 8. (Top) EPR spectra in CH₂Cl₂at 77 K of [(F₈)Fe^III]SbF₆(blue, g_^= 5.84) and (F₈)Fe^IIICl (red, g_^= 6.16). (Bottom) EPR spectra of

[(F₈)Fe^III]SbF₆in two solvents at 77 K, possessing g_^= 5.84 in CH₂Cl₂(blue) and g_^= 6.12 in THF (green) at 77 K.

Figure 9. ¹H NMR spectra of [(F₈)Fe^III(THF)₂]SbF₆in THF-d₈at various temperatures: þ20, -40, and -80 ꢀC from top to bottom.

N₂O_(g), analogous to one enzyme turnover of nitrogen

monoxide reactivity with certain CcO’s. The overall results

are not greatly altered from those obtained by instead using a

binuclear (⁶L)-heme/Cu complex (Scheme 2).¹Heme-Fe^II,

1418 Inorganic Chemistry, Vol. 49, No. 4, 2010

Wang et al.

Figure 10. ¹H NMR of the product mixture of the NOR reaction: {(F₈)Fe(NO)₂þ [(tmpa)Cu^I(MeCN)]^þþ 2 H^þ} in CD₃CN at RT.

Cu^I, and acid are all required to trigger the reaction. The

Scheme 4

dinitrosyl complex (F₈)Fe(NO)₂provides exactly 2 equiv of

•NO per heme-and-copper moiety, allowing us to monitor a

stoichiometric reaction. The dinitrosyl complex has been

characterized by variable-temperature NMR and UV-vis,

as well as EPR spectroscopies. DFT calculations by Ghosh

et al.^59,60and Ford et al.⁵⁸suggest that heme dinitrosyl

complexes possess a so-called “cis” conformation, which is

in accordance with the ¹H NMR spectroscopic data we

observe for (F₈)Fe(NO)₂. In addition to the five-coordinate

(F₈)Fe(NO) complex previously reported, a new six-coordi-

nate complex with THF as the axial base, (THF)(F₈)Fe(NO),

was synthesized and characterized by UV-vis, EPR, and IR

spectroscopies. Also, [(F₈)Fe^III]SbF₆, a new variation on

previously well-known complexes, was synthesized to help

us identify the product of the heme/Cu/•NO_(g)chemistry

described here, and this has been determined to exist in

solution as an admixture of high- and intermediate-spin

states.

Concerning heme/Cu •NO_(g)reductive coupling (bio)-

chemistry, recent biophysical studies and DFT calculations

from Varotsis and co-workers^2,13,91,92have led to the sugges-

tion of what is referred to as a trans mechanism (Scheme 4).

This was adopted on the basis of their biophysical and DFT

studies, with reference to the literature on heme/nonheme

diiron-containing NORs (see the Introduction). Rate-limit-

ing addition of •NO_(g)and a proton to heme-nitrosyl

complex II leads to a “N₂O₂H” intermediate which possesses

a N-N bond; note that reduced Cu^I_Bis proposed to be

present in this species. The oxidized and protonated hyponi-

trite (deprotonated hyponitrite is N₂O₂^2-, so III would

possess a protonated N₂O₂^-moiety, Scheme 4)) is proposed

to be N-bound to both the metal ions. These researchers

assert direct detection of intermediates II and III via reso-

nance Raman spectroscopic interrogation;^2,13,91however, the

latter hyponitrite complex was generated by adding excess

•NO_(g)to an oxidized (Fe^IIICu^II) form of the enzyme and

3 3 3

may be one-electron oxidized from intermediate III. Finally,

it is suggested that further protonation leads to N₂O genera-

tion, Scheme 4.

In light of this proposed enzyme mechanism (Scheme 4),

we suggest possible steps in the chemistry described in the

present report. A likely first step in the reaction would be the

attack of the copper(I) complex ([(tmpa)Cu^I(MeCN)]^þ) on

the nitrogen monoxide ligand in (F₈)Fe(NO)₂; an analogous

step probably also occurs when N₂O is produced from the

(⁶L)-heme/Cu system (Scheme 2). This may release one

•NO_(g)molecule, which now attacks

a binuclear

heme-Fe^II-(NO)-Cu^Ispecies to give something like inter-

mediate III (Scheme 4), that is, a hyponitrite-type species.

Protonation, electron-transfer from Cu^þof the (tmpa)Cu^I

moiety, and N-O cleavage could give N₂O_(g)and water, and

in our case the heme-Fe^IIIand Cu^IIcomplexes observed. We

suggest that there are other possibilities that should be

considered here, and for other synthetic systems to be studied

in the future, or even for the enzymes, see below.

(91) Pinakoulaki, E.; Ohta, T.; Soulimane, T.; Kitagawa, T.; Varotsis, C.

J. Am. Chem. Soc. 2005, 127, 15161–15167.

(92) Ohta, T.; Kitagawa, T.; Varotsis, C. Inorg. Chem. 2006, 45, 3187–

3190.

Article

Inorganic Chemistry, Vol. 49, No. 4, 2010 1419

The likelihood of N-N coupling occurring from a

is the role of iron versus copper in NO binding and stabiliza-

tion of a hyponitrite intermediate via O or N ligation? Does

electron transfer to two NO molecules occur from both

Fe-N-O O-N-Cu species would seem to us to be

3 3 3

problematic. Moenne-Loccoz et al.¹²recently suggested that

€

2-

transient binding of NO to the copper ion may occur as either

an O-bound (η¹-O) or a side-on (η²-NO) complex, as sup-

ported by FTIR and resonance Raman spectroscopic studies.

Such a situation could lead to a geometry or juxtaposition of

metals together to give ligated N₂O₂or a protonated

derivative? Or is the process stepwise, one-electron at a time,

as suggested in Scheme 4? What is the role and timing of

proton addition? How does an axial base (proximal histidine

to heme Fe) function during the whole process? Note that we

did not have a strong “base” for the heme in the present

system, nor for that based on the ⁶L heterobinuclear complex

reported earlier.¹For the proteins, Varotsis and co-workers

suggest that initial •NO_(g)binding to the ferrous heme leads

to Fe-N_histidinecleavage to give a five-coordinate heme-

nitrosyl.^11,91,94,95And of course, there are many basic ques-

tions to probe in heme/copper (or heme/nonheme diiron)

model systems concerning N-N bond formation and N-O

cleavage. We look forward to a rich coordination chemistry of

these binuclear heme/M systems and nitrogen oxide species.

the two NO moieties so as to greatly facilitate N-N forma-

12

€

tion. Further, Moenne-Loccoz et al. suggest that the

hyponitrite moiety is O-bound to both heme and copper

ion metal centers, contrary to what is suggested by Scheme 4.

Related to this hypothesis, Richter-Addo and co-workers⁹³

recently isolated a stable hyponitrite-bridged diiron(III)

complex (OEP)Fe-(trans-^-ONdNO^-)-Fe(OEP). An X-

ray structure revealed hyponitrite dianion O,O⁰ligation.

Protonation of this species led to Fe-O and O-N bond

cleavage and a release of N₂O_(g)

.

Thus, more efforts are needed to investigate the catalytic

mechanism of this •NO_(g)reductive coupling chemistry in

CcO’s. For this present system or analog synthetic systemswe

design in the future, investigations will be directed toward a

number of goals. We wish to find or stabilize a possible

hyponitrite or other intermediate(s) that may form and

deduce details of the coordination chemistry involved. What

Acknowledgment. This work was supported by a grant

from the National Institutes of Health (K.D.K., GM 60353)

and Korea WCU-Program R31-2008-000-10010-0 (K. D. K.).

Note Added after ASAP Publication. This article was

released on December 23, 2009, with errors in the Figure 6

caption and another minor text error. The correct version

was posted on January 11, 2010.

(93) Xu, N.; Campbell, A. L. O.; Powell, D. R.; Khandogin, J.;

Richter-Addo, G. B. J. Am. Chem. Soc. 2009, 131, 2460–2461.

(94) Stavrakis, S.; Pinakoulaki, E.; Urbani, A.; Varotsis, C. J. Phys.

Chem. B 2002, 106, 12860–12862.

(95) Pinakoulaki, E.; Stavrakis, S.; Urbani, A.; Varotsis, C. J. Am. Chem.

Soc. 2002, 124, 9378–9379.

Supporting Information Available: X-ray structure CIF file;

UV-vis, EPR, and NMR spectroscopic details. This material is

available free of charge via the Internet at http://pubs.acs.org.

Article Doi

Reductive coupling of nitrogen monoxide (?NO) facilitated by heme/copper complexes

DOI: 10.1021/ic901431r

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Article abstract of DOI:10.1021/ic901431r

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