Electron-Transfer Chemistry of the Iron-Molybdenum Cofactor Of
Total Page:16
File Type:pdf, Size:1020Kb
FULL PAPER Electron-Transfer Chemistry of the Iron ± Molybdenum Cofactor of Nitrogenase: Delocalized and Localized Reduced States of FeMoco which Allow Binding of Carbon Monoxide to Iron and Molybdenum Christopher J. Pickett,*[a] Kylie A. Vincent,[b] Saad K. Ibrahim,[a] Carol A. Gormal,[a] Barry E. Smith,[a] and Stephen P. Best*[b] Abstract: The electron-transfer chemis- fur core of the cofactor, whilst the third bands at 1885 and 1920 cmÀ1 respective- try of the isolated iron ± molybdenum irreversible process is localised on mo- ly. Moreover, in parallel with earlier cofactor of nitrogenase (FeMoco) has lybdenum. This is strongly reinforced by studies on the enzyme system, it is been studied by electrochemical and spectroelectrochemical studies under shown that at low CO concentration, spectroelectrochemical methods. Two 12CO and 13CO which reveal two inde- carbon monoxide binds to the cofactor interconverting forms of the cofactor pendent carbon monoxide binding sites in bridging modes, with n(CO) bands at arise from a redox-linked ligand isomer- that are specifically associated with the 1835 and 1808 cmÀ1 that are intercon- ism at the terminal iron atom;this is second (iron core) and third (molybde- verted by single-electron transfer. Impor- attributed to rotamerism of an anionic num) electron-transfer processes and tantly we show that the contentious over- N-methyl formamide ligand bound at which give rise to terminal n(12CO) all 2e difference in the assignment of the this site. FeMoco in its EPR-silent metal oxidation levels in the resting state oxidised state is shown to undergo three of the enzyme-bound cofactor, arising Keywords: bridging ligands ¥ cofac- successive one-electron transfer steps. from analysis of 57Fe ENDOR and Mˆss- tors ¥ electrochemistry ¥ IR We argue that the first and second redox bauer data, can be resolved in the light of spectroscopy ¥ nitrogen fixation ¥ processes are associated with electron- the electron-transfer chemistry of the nitrogenases transfer delocalised over the iron ± sul- isolated cofactor described herein. Introduction Integration of substrate binding with electron and proton transfer at an {Fe7S9Mo} cluster confined within a protein is the essence of a key biological process, nitrogen fixation.[1] The structure of this cluster (the M centre) within the crystalline resting-state nitrogenase MoFe protein has been established by Rees and co-workers and is represented in Figure 1.[2] However, the site and mode of binding of molecular nitrogen to the cluster and the mechanism of its subsequent reduction to ammonia are unknown. Consequent- ly, there has been considerable scope for speculation on how nitrogenase works with diverse mechanisms postulated from Figure 1. View of the M centre in the MoFe protein of A. vinelandii chemical and theoretical studies on model[3, 4] and in silico[5] nitrogenase from X-ray crystallographic data of Rees et al. as first reported in 1992.[2] [a] Prof. C. J. Pickett, Dr. S. K. Ibrahim, C. A. Gormal, Prof. B. E. Smith Department of Biological Chemistry, John Innes Centre systems. A very recent high-resolution structure of the MoFe Norwich, NR4 7UH (UK) protein of nitrogenase from Azotobacter vinelandii has led to Fax : (44)1603-450018 a revision of the nature of the M centre: it is suggested that an E-mail: [email protected] interstitial N atom (or possibly C or O) is contained within the [b] Dr. S. P. Best, K. A. Vincent central 6Fe3S cavity, octahedrally coordinated by the Fe School of Chemistry, University of Melbourne atoms. The presence of this light atom clearly disposes of the Victoria 3010 (Australia) Fax : (61)39347-5180 peculiar unsaturation of the six ™trigonal∫ iron atoms and is E-mail: [email protected] most likely a structural element cross-linking the atoms of the 76 ¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0947-6539/03/0901-0076 $ 20.00+.50/0 Chem. Eur. J. 2003, 9,No.1 76±87 trigonal prism to give a robust structure.[6] Key questions such the discussion, the corresponding states of FeMoco in the as the oxidation states of the metal atoms in the cluster and enzyme (the M centre) are designated Msemired,Mox and so the net charge it bears in the resting or other states remain forth. In the text, successive reduction processes of FeMocoox controversial as discussed below. There are as yet really no are labelled I, II, and III, and electrochemical parameters o' synthetic systems that adequately model structure and such as the formal potential E and peak current ip associated function of the enzyme, although the capacity of abiological with these processes are accordingly superscripted, fop mononuclear Mo or W systems to convert dinitrogen to example, IEo'. ammonia under ambient conditions is well established;[3, 7] chemical precedent for a carboxylate ligand functioning as a The state of ligation of FeMoco: The protein cysteinyl and leaving group to unmask a dinitrogen binding site has been histidine ligands are de-coordinated from FeMoco when it is demonstrated,[8] and NÀN bond cleavage reactions at dinu- extracted from the protein into NMF. The question arises as to clear metal sulfur centres described.[9] the nature of the exogenous ligands that coordinate to the The M centre is ligated at the terminal Fe atom by a terminal iron atom (Feterm) and of those that occupy the cysteinyl ligand and at the terminal Mo by a histidine ligand capping Mo coordination sites of the isolated cofactor. (Figure 1).[2] Rupturing this ligation allows extraction into N- methyl formamide (NMF) of the cofactor, FeMoco, which has The terminal iron site: Earlier IR studies indicate that the N- the ability to restore nitrogen-fixing activity to a mutant deprotonated NMF anion ligates the terminal Fe atom,[18] protein devoid of the cluster.[10] Comparison between the whereas sulfur K X-ray absorption edge studies (XANES) EXAFS, EPR and Mˆssbauer spectroscopy of isolated have led to the proposal that Feterm is ligated by a thiosulfate FeMoco and that of the native protein, together with ligand.[19] analytical data, indicate that the {Fe7S9Mo} cluster core is Figure 3 (lower trace) shows the cyclic voltammetric conserved in the extracted cofactor and that it retains the behaviour of FeMocoox in the potential domain which chelating homocitrate ligand (Figure 2).[11] encompasses the primary reduction process that gen- erates the FeMocosemired system. The system displays two S - O2CH2C CH CH CO - S S 2 2 2 O Fe Fe O X Fe S Fe X Fe S Mo O S Fe Fe L S S S Figure 2. Schematic structure of isolated FeMoco. The interstitial light atom X is N (or possibly C or O) according to a recent high-resolution MoFe protein crystallographic analysis.[6] Protocols for obtaining NMF solutions of FeMoco at ca. 1mm concentration from the MoFe protein of nitrogenase in sufficient quantity are now well established. This has provided the opportunity for a wide range of direct chemical, spectro- Figure 3. Lower trace: Cyclic voltammetry of a solution of FeMocoox in scopic, kinetic and electrochemical studies of the cofactor; NMF at a vitreous carbon electrode recorded at 50 mV sÀ1 over a potential these complement studies on the whole enzyme system and range encompassing the primary reduction process, showing two redox- are beginning to provide some insights into how exogenous dependent isomer forms of FeMocoox/semired. Upper trace: Primary and ox À1 ligands, substrates and inhibitors interact with the isolated secondary reductions of FeMoco(SPh) at 300 mVs . The double-headed arrows represent one-electron currents based on Ii red. cluster.[12±15] p In this paper we describe aspects of the electrochemistry of FeMoco isolated from wild-type Klebsiella pneumoniae nitro- major redox active forms. The scan rate dependence of the genase. Seminal work on the electrochemistry of FeMoco from voltammetry confirms that the redox couples are intercon- Azotobacter vinlandii was described several years ago by verted in dynamic equilibria as observed by Schultz and co- Schultz and co-workers,[16] and we take their studies as our workers.[20] The difference in potential between the two redox starting point. As expected from crystallographic data,[17] couples is relatively small (90 mV);this is indicative of FeMoco isolated from either K. pneumoniae or A. vinlandii geometric or linkage isomerism rather than gross structural has indistiguishable spectroscopic and electrochemical proper- change. The structural integrity of the {Fe7S9Mo} core upon ties. extraction of the cofactor indicates that the redox isomer- isation is associated with one or other of the exogenous ligand Results and Discussion sites. The redox isomerism collapses when one equivalent of thiophenolate anion is added to the system and it is replaced Herein the S 3/2 semireduced and EPR silent one-electron by a single reversible one-electron process, with Eo' close to oxidised states of the extracted cofactor are abbreviated as that of the high-potential isomeric couple.[8, 12] Cysteine FeMocosemired and FeMocoox, respectively;other accessible methyl ester similarly causes the collapse of the redox redox states are correspondingly superscripted in the text. In isomerism. Chem. Eur. J. 2003, 9, No. 1 ¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0947-6539/03/0901-0077 $ 20.00+.50/0 77 FULL PAPER C. J. Pickett, S. P. Best et al. The natural ligation of the Feterm in the MoFe protein of ment. The equilibrium distribution of the isomers depends nitrogenase is the thiolate group of a cysteinyl residue, and upon the redox state of the complex with reduction favouring EXAFS studies of the isolated cofactor are concordant with rotation of the donor oxygen atom away from the metal [21] [23] thiolates similarly binding to Feterm . Clearly the loss of the centre.