A functional nitric oxide reductase modelJames P. Collman*, Ying Yang, Abhishek Dey, Richard A. Decreau, Somdatta Ghosh,Takehiro Ohta, and Edward I. Solomon

Department of Chemistry, Stanford University, Stanford, CA 94305

Contributed by James P. Collman, September 2, 2008 (sent for review August 7, 2008)

A functional heme/nonheme nitric oxide reductase (NOR) model ispresented. The fully reduced diiron compound reacts with twoequivalents of NO leading to the formation of one equivalent ofN2O and the bis-ferric product. NO binds to both heme Fe andnonheme Fe complexes forming individual ferrous nitrosyl species.The mixed-valence species with an oxidized heme and a reducednonheme FeB does not show NO reduction activity. These resultsare consistent with a so-called ‘‘trans’’ mechanism for the reduc-tion of NO by bacterial NOR.

functional model � NO reduction � N2O � “trans” mechanism

N itric oxide reductase (NOR) is a membrane-bound enzymethat catalyzes the 2e� reduction of nitric oxide (NO) to

nitrous oxide (N2O), an obligatory step involved in the sequen-tial reduction of nitrate to dinitrogen known as bacterial deni-trification. The active site of NOR consists of a monohistidineligated five-coordinate heme and a trisimidazole ligated non-heme FeB. This structure strongly resembles the active site ofoxygen reduction enzyme-cytochrome c oxidase (CcO), whichpossesses a heme-a3/CuB center (Fig. 1) (1, 2). Essentially, thedistal metal CuB in CcO is replaced by a nonheme Fe metal inNOR; NOR and CcO are thought to be distant relatives.

The dinuclear iron active site in NOR was confirmed a decadeago by spectroscopic studies (3). Presumably, two NO moleculesare turned over to give one molecule of N2O and one moleculeof H2O at the diiron center with the consumption of twoelectrons and two protons. Although many enzyme studies ofNOR have been focused on the intermediate trapping andelucidation of the reaction mechanism (4–15), the details of thecatalytic cycle are still unresolved because of the lack ofstructural information and uncertainty regarding short-livedintermediates.

In contrast to enzyme studies, synthetic biomimetic modelcomplexes provide a straightforward and controlled method tounderstand how this chemical transformation proceeds at theenzyme active site. However, only a few synthetic models havebeen developed that mimic the active site of NOR; moreover,these compounds either lack a proximal imidazole ligand (16, 17)or use pyridine as a replacement for the histidine ligands(18–20). No functional NOR models have been reported to date.Our CcO model complexes have proved to be functionally activefor oxygen reduction reaction with minimal reactive oxygenspecies (ROS) formation (21–24). These appear to be promisingNOR model candidates if the distal Cu metal is replaced by aniron because the resulting diiron compound has almost all of thekey components in NOR: a heme Fe with a proximal imidazoleligand and a trisimidazole ligated nonheme Fe center.

In this report, we disclose the first synthetic functional NORmodel LFeII/FeII (Fig. 2), which reacts with two equivalents ofNO to give one equivalent of N2O and the bis-ferric product. Wehave shown that NO binds to both heme Fe and FeB to form apossible bis-nitrosyl intermediate; subsequently, the two boundNO molecules are reduced to N2O by electrons from both Fecenters, leaving both heme Fe and FeB in an uncoupled ferricstate. N2O has been quantitatively identified by using an enzyme,nitrous oxide reductase (N2OR) that reduces N2O to N2. Thebis-ferric product was characterized by both FTIR and EPR:

FTIR reveals a heme ferric nitrosyl band at 1,924 cm�1 that shiftsto 1,887 cm�1 when 15NO is used, whereas the EPR spectrummanifests a low-spin ferric signal that is assigned to the nonhemeFeB because a ferric heme nitrosyl would be EPR silent. The NOadducts of both heme Fe and FeB were obtained separately fromthe reaction of NO with LFeII and LZnII/FeII. These NO adductswere characterized with a series of spectroscopic methods in-cluding UV-vis, EPR, resonance Raman, FTIR, and mass spec-troscopy. The reaction of NO with the mixed-valence compoundLFeIII/FeII was also investigated. Our data show the formation ofa mixture of LFeII-NO/FeIII and LFeIII-NO/FeII-NO species, butno N2O was detected. These results are consistent with aso-called ‘‘trans’’ mechanism for the reduction of NO to N2O byNOR.

ResultsSyntheses and Characterization of Dinuclear Complexes. The re-duced diiron complex LFeII/FeII is readily synthesized by reactionof LFeII complex (25) with 1 equivalent of Fe(OTf)2(MeCN)2 inTHF at room temperature under a N2 atmosphere. The UV-visspectrum shows a fast and clean formation of a dinuclear productwith shifts for both the Soret (426–424 nm) and Q bands(535–530 nm) and a slightly diminished intensity in both bands(Fig. 3, solid to dotted line). The dinuclear compound LZnII/FeII

was synthesized by using a similar method from reaction of LZnII

with Fe(OTf)2(MeCN)2 [UV-vis spectra are in supporting in-formation (SI) Fig. S1]. The mixed-valence compound LFeIII/FeII was obtained by oxidation of LFeII/FeII with one equivalentof ferrocenium tetrafluoroborate. The bis-ferric LFeIII/FeIII wasgenerated by oxidation of LFeII/FeII with either two equivalentsof ferrocenium tetrafluoroborate or with dioxygen. The struc-tures of these dinuclear compounds were confirmed by electro-spray mass spectroscopy.

Author contributions: J.P.C. and Y.Y. designed research; Y.Y., A.D., R.A.D., S.G., and T.O.performed research; Y.Y. contributed new reagents/analytic tools; Y.Y., A.D., R.A.D., S.G.,T.O., and E.I.S. analyzed data; and Y.Y. and A.D. wrote the paper.

The authors declare no conflict of interest.

*To whom correspondence should be addressed. E-mail: jpc@stanford.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808606105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

Fig. 1. Schematic representation of the bimetallic active sites of NOR andCcO.

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Reaction of NO with LFeII, LZnII/FeII and LFeII/FeII. LFeII � NO. Additionof purified NO to a THF solution of LFeII leads to formation ofits mono-nitrosyl derivative LFe-NO. This reaction is associatedwith a decreased intensity of the Soret band at 427 nm and a Qband shift from 535 to 550 nm (Fig. 4). The EPR spectrum showsa S � 1/2 signal (Fig. 8, solid line) typical of a six-coordinateferrous NO complex akin to those reported for the active site inCcO (26–30). The FTIR spectrum shows a new band at 1,630cm�1 for the LFe-NO complex that shifts to 1,600 cm�1 with15NO (Fig. 5, dotted and dashed lines). The resonance Ramanspectrum obtained by excitation with a 425-nm laser shows anFe-N stretch at 581 cm�1 that shifts to 545 cm�1 with 15NOsubstitution. An Fe-NImidazole stretch is also observed at 238cm�1, providing further evidence for the presence of a six-coordinate iron nitrosyl (Fig. 6).LZnII/FeII�NO. Addition of NO to LZnII/FeII in THF results inslight blue shifts (0.5 nm for the Soret band and 1 nm for the Qband) in the UV-vis spectra (Fig. S1). The FTIR of the 14NOadduct of LZnII/FeII exhibits a new vibration frequency at 1,810cm�1 (Fig. 7, dotted line) that shifts to 1,774 cm�1 with 15NOsubstitution (Fig. 7, dashed line). These features are not ob-served in the LZnII/FeII (Fig. 7, solid line). The Fe-NO vibrationscould not be identified with resonance Raman because thisregion was obscured by porphyrin bands. The EPR spectrum

reveals a well characterized S � 3/2 nonheme Fe-NO signal atg � 4.0 (Fig. 8, dashed line). Note that the small perturbation ofthe Zn-porphyrin Soret implies that the NO probably binds in thepocket but does not bridge the two metals. Binding of NO to FeBleads to LZnII/FeII-NO species. Both the LFeII-NO and LZnII/FeII-NO species are stable in the solid state for a prolongedperiod when exposed in air. These mononitrosyl complexes werealso characterized by high-resolution electrospray mass spec-troscopy (Figs. S2–S5).LFeII/FeII�NO. Addition of NO to the THF solution of LFeII/FeII

at room temperature leads to rapid changes in the UV-visspectrum. The Soret band shifts from 424 to 423 nm, and the Qband shifts from 530 to 550 nm (Fig. 3, dotted to dashed line).FTIR spectra obtained from solid samples of the end productexhibit a new band at 1,924 cm�1 that is absent in LFeII/FeII (Fig.9, dotted and solid lines). This band is shifted to 1,887 cm�1 when15NO is used for the sample preparation (Fig. 9, dashed line).The 37-cm�1 15N-isotope shift is consistent with literature datafor heme ferric nitrosyls (18, 31, 32). The resonance Raman dataon this complex show a Fe-NO stretch at 610 cm�1 (Fig. 10, solidline) that shifts to 598 cm�1 upon 15NO substitution (Fig. 10,dotted line). The Fe-NO bending vibration of this species isobserved at 589 cm�1 that shifts to 580 cm�1 upon 15NOsubstitution. These values are characteristic of a ferric heme NOspecies. The EPR data of the end product show an S � 1/2 signal(g � 2.07, 2.02, 1.96) that is not perturbed by 15NO substitution(Fig. 8, dotted line). These data and spin integration indicate thata single low-spin FeIII is present in the product. The abovespectroscopic data are consistent with the formation of LFeIII-NO/FeIII-OH (Scheme 1); the vibrational features i.e., Fe-N �610 cm�1 in the Raman and N-O � 1,924 cm�1 in the FTIR arederived from a ferric heme nitrosyl that is diamagnetic (i.e., EPR

Fig. 2. Representation of the model complexes of ligand L: LFeII (no MB);LFeII/FeII (M � MB � Fe2�); LZnII/FeII (M � Zn2�, MB � Fe2�); LFeIII/FeII (M � Fe3�,MB � Fe2�); LFeIII/FeIII (M � Fe3�, MB � Fe3�) (metals have triflate counterions).

Fig. 3. UV-vis spectra of LFeII (solid line), LFeII/FeII (dotted line), and LFeII/FeII�NO (dashed line).

Fig. 4. UV-vis spectra of LFeII (solid line) and LFeII�NO (dotted line).

Fig. 5. IR spectra of LFeII (solid line) and its 14NO (dotted line) and 15NO(dashed line) derivatives.

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silent), and the S � 1/2 EPR signal is derived from the nonhemeferric center in the distal pocket. The assignment of this endproduct is further buttressed by comparable FTIR, resonanceRaman, and EPR (g � 2.07, 2.00, 1.95) obtained by addition ofNO to LFeIII/FeIII, followed by addition of an equivalent ofsodium methoxide (comparable to OH) (Fig. S6).†

We have used a nitrous oxide reductase enzyme (N2OR) thatreduces N2O to N2 to identify the formation of N2O in thisreaction. Samples with 1 mM LFeII/FeII on addition of 3 mM NOshow specific activities of 43 � 5, whereas the backgroundactivity with the same amount of NO gas is �10 � 2. This activityreflects an N2O concentration of 1 mM in solution, which, in thiscase, implies a quantitative yield. This, in addition to thevibrational and EPR data presented above, indicates that thesynthetic model complex LFeII/FeII reduces two molecules of NOto N2O (Scheme 1).

Reaction of NO with the Mixed-Valence Compound LFeIII/FeII. Addi-tion of NO to the mixed-valence compound LFeIII/FeII shifts theSoret from 418 to 424 nm and the Q band from 530 to 546 nm(Fig. 11). The FTIR of the solid samples from the reactionmixture reveals a band at 1,924 cm�1, which indicates theformation of a ferric heme nitrosyl species, and another weakband at 1,812 cm�1, which is indicative of a ferrous nonhemenitrosyl species (Fig. S7). EPR data from the NO adduct withLFeIII/FeII indicates a mixture of a heme Fe-NO (signal at g �2.0), a nonheme Fe-NO species (signal at g � 4.0), and ahigh-spin FeIII species (at g � 6) (Table 1). This demonstratesthat the product of LFeIII/FeII�NO is a mixture of LFeIII-NO/FeII-NO and LFeII-NO/FeIII. The ferric heme nitrosyl is EPRsilent, but an N-O stretch is observed in the IR. This implies thatNO binding to the LFeIII/FeII complex leads to some electrontransfer from the ferrous nonheme to the ferric heme center,resulting in an equilibrium of three iron nitrosyl species: ferricheme nitrosyl, ferrous heme nitrosyl, and ferrous nonhemenitrosyl. In any case, these mixed-valence species do not formN2O.

DiscussionThe Synthetic Heme/Nonheme Diiron Compound Is a Functional NORModel. In this study, a synthetic model compound is reported thatintegrates the essential features proposed for a bacterial NORactive site; namely, a heme active site with a covalently attachedimidazole ligand and a nonheme site coordinated via threeimidazole ligands. This model possesses the functional NO

reductase activity i.e., two molecules of NO are reduced to onemolecule of N2O at the fully reduced diiron center, leaving adi-ferric compound. The yield of N2O is nearly quantitativewithin the error of the enzyme assay. A putative diferric complexformed initially after reduction of NO to N2O reacts further withNO, forming a Fe3�-NO/Fe3�-OH species as indicated by EPR,FTIR, and resonance Raman spectroscopy methods. The�(N-O) of our ferric heme nitrosyl is �20 cm�1 higher than the�(N-O) of the ferric heme nitrosyl of NOR (1,904 cm�1) (33) andcytochrome cbb3 oxidase (1,903 cm�1) (34) but similar to theneutral Met Mb-NO (1,921 cm�1) (31, 35). The EPR spectrumof the reaction product reveals a low-spin ferric signal, which isassigned to a ferric nonheme because a ferric heme nitrosylwould be EPR silent (S � 0). A bis-ferric compound LFeIII-NO/FeIII-OMe prepared by the reaction of LFeIII/FeIII with NO andone equivalent of NaOMe exhibits a very similar low-spin ferricsignal in the EPR. This strongly suggests that an OH ligand isbound to the ferric nonheme center (Table 1).

Mechanism of NO Reduction. The molecular mechanism of the NOreduction by NOR is still under debate. Two mechanisms havebeen proposed for binding and reduction of NO at the active site:A “trans” mechanism invokes binding of two NO molecules tothe heme-iron and nonheme iron separately at the fully reducedactive site, whereas a ‘‘cis’’ mechanism suggests binding of bothNO molecules to only one iron metal (typically FeB). Recentquick-freezing EPR (15) indicates the formation of both heme-iron nitrosyl and nonheme-iron nitrosyl species, supporting the“trans” mechanism. Meanwhile, the mixed-valent state (heme-FeIII/FeB

II) because of the low midpoint potential of heme b3(heme b3, Em � 60 mV; FeB, Em � 320 mV), was suggested tobe the active form of the enzyme (10). Therefore, it had beenproposed that the binding and reduction of NO occurs exclu-sively on FeB, leaving the heme Fe uninvolved during the

†The reaction of LFeIII with NO led to the formation of LFeIII-NO, which is characterized byUV-vis, IR, and resonance Raman; no reduction of ferric heme to form heme Fe(II)-NO wasobserved.

Fig. 6. Resonance Raman data on LFe-14NO (solid line) and LFe-15NO (dottedline).

Fig. 7. IR spectra of LZnII/FeII (solid line) and its 14NO (dotted line) and 15NO(dashed line) derivatives.

Fig. 8. EPR data of the LFeII-NO (solid line), LZnII/FeII-NO (dotted line), andLFeII/FeII�NO (dotted-to-dashed line) complexes.

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catalytic process (10, 11). In addition, it has also been suggestedthat two molecules of NO bind consecutively at the heme Fe3�

site to form a hyponitrite intermediate, which is thought todecay, giving the ferric heme, N2O, and H2O. A six-coordinateheme Fe3�-NO species of cytochrome cbb3 oxidase and ahyponitrite species in the Fe-Cu dinuclear center were detectedby FTIR and the resonance Raman spectroscopy method, re-spectively (34, 36).

We have shown that NO can readily bind to heme Fe andnonheme Fe separately to form stable heme Fe-NO and non-heme Fe-NO species. This indicates that the enzymatic reactionprobably proceeds by a “trans” mechanism. Two equivalents ofNO react with the diiron center and form individual heme andnonheme nitrosyl species. Then, the two adjacent bound ni-trosyls undergo reductive coupling to form N2O (possiblythrough a Fe-N(O)-N(O)-Fe intermediate), leaving both theheme Fe and the nonheme Fe in an oxidized ferric state. It is notclear if a �-oxo diiron compound is formed as an intermediateduring this reaction, because excess NO may bind to the hemeFe and cause the rupture of Fe-O-Fe bond, leading to theobserved final LFeIII-NO/FeIII-OH compound. The reactionproduct of LFeII/FeII with two equivalents of NO did not showa ferric heme nitrosyl signal in the FTIR, and a small ion peakat m/z 1,491 amu corresponding to a [LFe-O-Fe(MeCN)2]� wasdetected by electrospray mass spectroscopy.

It has been postulated that NO binding to the heme iron ofNOR or CcO causes bond cleavage between heme Fe and theproximal imidazole producing a five-coordinate heme nitrosylcomplex (5, 8). However, we show that binding NO to LFeII

results in a stable six-coordinate heme nitrosyl in which the

proximal imidazole is still coordinated to the heme iron. Such asix-coordinate ferrous nitrosyl does not appear to be a ‘‘dead-end’’ species as previously claimed (10). Instead, the boundnitrosyl can undergo N-N coupling with the adjacent nonhemenitrosyl to produce N2O. Praneeth et al. (37) demonstrated thata six-coordinate iron(II) porphyrin NO adduct with a proximalimidazole ligand has a distinctive FeIINO� character relative to afive-coordinate FeII-NO compound (37). Moreover, the en-hanced radical character of the heme nitrosyl should be advan-tageous for the central (radical) N-N coupling step in the “trans”mechanism. On the other hand, a less reactive five-coordinateFeII-NO would be a dead-end species, as demonstrated by arecent synthetic diiron model based on a five-coordinate hemenitrosyl, which does not show any NO reductase activity (18).

The Mixed-Valence Form of NOR Is Not Active for the Reduction of NOto N2O. It has been suggested that NO activation occurs with amixed-valence form of the NO reductase with an oxidized heme b3and a reduced nonheme FeB (10). Based on this, it was proposedthat the binding of two molecules of NO happens exclusively oneither heme Fe or FeB, leaving the other metal essentially a witness.However, our studies demonstrated that reaction of a mixed-valence compound LFeIII/FeII with NO leads to a mixture of twospecies: LFeIII-NO/FeII-NO and LFeII-NO/FeIII. No N2O was

Fig. 9. FTIR spectra of LFeII/FeII (solid line), LFeII/FeII �14NO (dotted line), andLFeII/FeII �15NO (dashed line)

Fig. 10. Resonance Raman of the reaction product of LFeII/FeII�NO with14NO (solid line) and 15NO (dotted line).

Fig. 11. The optical spectra of LFeIII/FeII (solid line) and LFeIII/FeII � NO(dotted line)

Scheme 1. Reactions of mono- and bis-iron complexes with NO. (A) LFeII �NO. (B) LZnII/FeII � NO. (C) LFeIII/FeII � NO. (D) LFeII/FeII � NO.

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detected from either of these species, suggesting that a so-called‘‘cis’’ mechanism is unlikely in the NO reduction by NOR.

ConclusionsWe have described a functional heme/nonheme nitric oxidereductase model LFeII/FeII that can reduce NO to N2O stoichio-metrically, leading to a bis-ferric product. NO binds to the hemeFe of LFeII, producing a stable six-coordinate heme Fe-NOcomplex, whereas binding of NO to a nonheme Fe of LZnII/FeII

leads to a nonheme Fe-NO species. These results suggest that thereaction of LFeII/FeII with NO follows a “trans” mechanism: Twomolecules of NO bind to heme Fe and nonheme Fe separately,forming a heme Fe-NO and a nonheme Fe-NO species; then thetwo adjacent nitrosyls undergo reductive coupling, producingN2O and the di-ferric product. The mixed-valence form of ourmodel compound LFeIII/FeII does not show any NO reductionactivity; instead, stable nitrosylated species were formed. Ex-periments focusing on the reaction intermediates to furtherclarify the reaction mechanism await completion.

Materials and MethodsAll reagents were obtained from commercial suppliers and used withoutfurther purification unless otherwise indicated. Fe(OTf)2(MeCN)2 was pre-pared according to literature procedures (38). Heme compound LFeII wassynthesized as reported (25). All air- and moisture-sensitive reactions werecarried out under a nitrogen atmosphere in oven-dried glassware. Acetoni-trile, tetrahydrofuran, and dichloromethane were purified and dried by pass-ing reagent-grade solvent through a series of two activated alumina columnsunder nitrogen atmosphere. These solvents were further deoxygenated bybubbling with nitrogen for 30 min in a nitrogen glove box. DMF was distilledover molecular sieves and properly deoxygenated. Nitric oxide (NO) wasobtained from Matheson Gas Products or generated by adding saturatedNaNO2 solution into a sulfuric acid solution (98% sulfuric acid /water � 3:1). Itis purified by passage through a series of two thoroughly degassed 3.0 M KOHsolutions and water. The saturated NO solution in acetonitrile or THF wasmade by bubbling purified NO gas through deoxygenated solvent in a gas-tight vial for 15 min. All reactions with NO (including making EPR and UV-vissamples) were carried out by injecting saturated NO solution into the samplesolution in a glovebag purged and filled with nitrogen. The 15NO (99%) waspurified by passing through a column packed with dry KOH powder under N2.

Infrared spectra were obtained on a Mattson Galaxy 4030 FT-IR spectrom-eter. Solid samples were prepared by dissolving a sample in solution in a

glovebox, spotting on a KBr or NaCl palate, allowing the solvent to evaporate,and then covering it by another palate and sealing the sides with parafilm. Thepalates containing the sample were sealed in a container and brought to theIR spectrometer for measurement. Room-temperature UV-vis spectra wererecorded with a HP8452 diode array spectrophotometer. Mass spectra wereobtained from the Stanford Mass Spectrometry Laboratory. The air-sensitivesample solutions were prepared in a glovebox and sealed in gas-tight vials.They were brought to the spectrometer and injected into the instrumentimmediately before the measurement.

EPR spectra were obtained by using a Bruker EMX spectrometer, ER 041 XGmicrowave bridge, and ER 4102ST cavity. All X band samples were run at 77 Kin a liquid nitrogen finger dewar. A Cu standard (1.0 mM CuSO4�5H2O with 2mM HCl and 2 M NaClO4) was used for spin quantitation of the EPR spectra.

Resonance Raman (rR) spectra were obtained by using a Princeton Instru-ments ST-135 back-illuminated CCD detector on a Spex 1877 CP triple mono-chromator with 1,200, 1,800, and 2,400 grooves per millimeter holographicspectrograph gratings. Excitation was provided by a Dye Laser (Stilbene 599;Coherent) that was energized by a Coherent Innova Sabre 25/7 Ar� CW ionlaser. The laser line 425 nm (�10 mW) was used for excitation. The spectralresolution was �2 cm�1. Sample concentrations were �1 mM in Fe. Thesamples were either cooled to 77 K in a quartz liquid nitrogen finger dewar(Wilmad) and hand spun to minimize sample decomposition during scancollection or cooled to 190–196 K by using a flow of liquid-N2-cooled He gasin a spinner setup.

In evaluating for the possibility of generation of N2O in the reactionmixture, the reaction of LFeII/FeII with purified NO was performed in dichlo-romethane, and a buffer solution was used to extract this organic layer andthen used for the activity assay. Simultaneously, PnN2OR enzyme was incu-bated in an excess of an anaerobic solution of methyl viologen and dithionitein Tris buffer (pH �7.3), in the glovebox (required to activate the enzyme) (39).Activity of the enzyme was determined spectrophotometrically, after theoxidation of dithionite reduced methyl viologen at 600 nm by using a standardprotocol under anaerobic conditions (40, 41). The activity initiated by adding20 �l of the 100-�l buffer solution used to extract the reaction mixture inCH2Cl2 was 43 �mol of N2O reduced min�1mg�1 of N2OR. As a control, activitymeasured by initiating the reaction with NO-saturated buffer solution was 10units. A N2O concentration-vs.-activity curve shows that 43 units of activitycorrespond to an N2O concentration of 1 mM.

ACKNOWLEDGMENTS. We thank Dr. Allis Chien of the Stanford UniversityMass Spectrometry Group for mass spectrometry analysis. This material isbased on work supported by National Institutes of Health Grant GM-69658 (toJ.P.C.) and National Science Foundation Grant DMB0342807 (to E.I.S.). R.A.D.is thankful for a Lavoisier Fellowship.

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Table 1. Summary of spectroscopic features of LFeII-NO, LZnII/FeII-NO, LFeII/FeII�NO, and LFeIII/FeIII-OMe � NO

Spectroscopymethod LFeII-NO LZnII/FeII-NO LFeII/FeII�NO LFeIII/FeIII�NO LFeIII/FeII�NO

IR 14NO/ 15NO (cm�1) 1,630/1,600 1,810/1,774 1,924/1,887 1,924/1,885 1,924, 1,812 (14NO)EPR S � 1/2, g � 2.08,

2.02, 1.9714NAy�22 cm�1

15NAy�31 cm�1

S � 3/2 g � 4.0 S � 1/2 g � 2.07, 2.02, 1.96 S � 1/2, g � 6 afterreaction of NaOMe

(1 eq): g � 2.07,2.00, 1.95

g � 2.0 g � 4.0 g � 6

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