X-ray absorption and resonance Raman studies of methyl-coenzyme M reductase indicating that ligand exchange and macrocycle reduction accompany reductive activation

Methyl-coenzyme M reductase (MCR) catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). MCR contains a nickel hydrocorphin cofactor at its active site, called cofactor F(430). Here we present evidence that the macrocyclic ligand participates in the redox chemistry involved in catalysis. The active form of MCR, the red1 state, is generated by reducing another spectroscopically distinct form called ox1 with titanium(III) citrate. Previous electron paramagnetic resonance (EPR) and (14)N electron nuclear double resonance (ENDOR) studies indicate that both the ox1 and red1 states are best described as formally Ni(I) species on the basis of the character of the orbital containing the spin in the two EPR-active species. Herein, X-ray absorption spectroscopic (XAS) and resonance Raman (RR) studies are reported for the inactive (EPR-silent) forms and the red1 and ox1 states of MCR. RR spectra are also reported for isolated cofactor F(430) in the reduced, resting, and oxidized states; selected RR data are reported for the (15)N and (64)Ni isotopomers of the cofactor, both in the intact enzyme and in solution. Small Ni K-edge energy shifts indicate that minimal electron density changes occur at the Ni center during redox cycling of the enzyme. Titrations with Ti(III) indicate a 3-electron reduction of free cofactor F(430) to generate a stable Ni(I) state and a 2-electron reduction of Ni(I)-ox1 to Ni(I)-red1. Analyses of the XANES and EXAFS data reveal that both the ox1 and red1 forms are best described as hexacoordinate and that the main difference between ox1 and red1 is the absence of an axial thiolate ligand in the red1 state. The RR data indicate that cofactor F(430) undergoes a significant conformational change when it binds to MCR. Furthermore, the vibrational characteristics of the ox1 state and red1 states are significantly different, especially in hydrocorphin ring modes with appreciable C=N stretching character. It is proposed that these differences arise from a 2-electron reduction of the hydrocorphin ring upon conversion to the red1 form. Presumably, the ring-reduction and ligand-exchange reactions reported herein underlie the enhanced activity of MCR(red1), the only form of MCR that can react productively with the methyl group of methyl-SCoM.     

Direct determination of the number of electrons needed to reduce coenzyme F430 pentamethyl ester to the Ni(I) species exhibiting the electron paramagnetic resonance and ultraviolet-visible spectra characteristic for the MCR(red1) state of methyl-coenzyme M reductase

The UV-visible and electron paramagnetic resonance (EPR) spectra of MCR(red1), the catalytically active state of methyl-coenzyme M reductase, are almost identical to those observed when free coenzyme F430 or its pentamethyl ester (F430M) are reduced to the Ni(I) valence state. Investigations and proposals concerning the catalytic mechanism of MCR were therefore based on MCR(red1) containing Ni(I)F430 until, in a recent report, Tang et al. (J. Am. Chem. Soc. 2002, 124, 13242) interpreted their resonance Raman data and titration experiments as indicating that, in MCR(red1), coenzyme F430 is not only reduced at the nickel center but at one of the C=N double bonds of the hydrocorphinoid macrocycle as well. To resolve this contradiction, we have investigated the stoichiometry of the reduction of coenzyme F430 pentamethyl ester (F430M) by three independent methods. Spectroelectrochemistry showed clean reduction to a single product that exhibits the UV-vis spectrum typical for MCR(red1). In three bulk electrolysis experiments, 0.96 +/- 0.1 F/mol was required to generate the reduced species. Reduction with decamethylcobaltocene in tetrahydrofuran (THF) consumed 1 mol of (Cp)(2)Co/mol of F430M, and the stoichiometry of the reoxidation of the reduced form with the two-electron oxidant methylene blue was 0.46 +/- 0.05 mol of methylene blue/mol of reduced F430M. These experiments demonstrate that the reduction of coenzyme F430M to the species having almost identical UV-vis and EPR spectra as MCR(red1) is a one-electron process and therefore inconsistent with a reduction of the macrocycle chromophore.     

Nickel oxidation states of F(430) cofactor in methyl-coenzyme M reductase

Magnetic circular dichroism (MCD) spectroscopy and variable-temperature variable-field MCD are used in combination with density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations to characterize the so-called ox1-silent, red1, and ox1 forms of the Ni-containing cofactor F430 in methyl-coenzyme M reductase (MCR). Previous studies concluded that the ox1 state, which is the precursor of the key reactive red1 state of MCR, is a Ni(I) species that derives from one-electron reduction of the Ni(II)-containing ox1-silent state. However, our absorption and MCD data provide compelling evidence that ox1 is actually a Ni(II) species. In support of this proposal, our DFT and TD-DFT calculations indicate that addition of an electron to the ox1-silent state leads to formation of a hydrocorphin anion radical rather than a Ni(I) center. These results and biochemical evidence suggest that ox1 is more oxidized than red1, which prompted us to test a new model for ox1 in which the ox1-silent species is oxidized by one electron to form a thiyl radical derived from coenzyme M that couples antiferromagnetically to the Ni(II) ion. This alternative ox1 model, formally corresponding to a Ni(III)/thiolate resonance form but with predicted S = 1/2 EPR parameters reminiscent of a Ni(I) (3dx2-y2)1 species, rationalizes the requirement for reduction of ox1 to yield the red1 species and the seemingly incongruent EPR and electronic spectra of the ox1 state.     

A nickel hydride complex in the active site of methyl-coenzyme m reductase: implications for the catalytic cycle

Methanogenic archaea utilize a specific pathway in their metabolism, converting C1 substrates (i.e., CO2) or acetate to methane and thereby providing energy for the cell. Methyl-coenzyme M reductase (MCR) catalyzes the key step in the process, namely methyl-coenzyme M (CH3-S-CoM) plus coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. The active site of MCR contains the nickel porphinoid F430. We report here on the coordinated ligands of the two paramagnetic MCR red2 states, induced when HS-CoM (a reversible competitive inhibitor) and the second substrate HS-CoB or its analogue CH3-S-CoB are added to the enzyme in the active MCR red1 state (Ni(I)F430). Continuous wave and pulse EPR spectroscopy are used to show that the MCR red2a state exhibits a very large proton hyperfine interaction with principal values A((1)H) = [-43,-42,-5] MHz and thus represents formally a Ni(III)F430 hydride complex formed by oxidative addition to Ni(I). In view of the known ability of nickel hydrides to activate methane, and the growing body of evidence for the involvement of MCR in "reverse" methanogenesis (anaerobic oxidation of methane), we believe that the nickel hydride complex reported here could play a key role in helping to understand both the mechanism of "reverse" and "forward" methanogenesis.     

Square planar vs tetrahedral coordination in diamagnetic complexes of nickel(II) containing two bidentate pi-radical monoanions

The reaction of three different 1-phenyl and 1,4-diphenyl substituted S-methylisothiosemicarbazides, H(2)[L(1-6)], with Ni(OAc)(2).4H(2)O in ethanol in the presence of air yields six four-coordinate species [Ni(L(1-6)(*))(2)] (1-6) where (L(1-6)(*))(1-) represent the monoanionic pi-radical forms. The crystal structures of the nickel complexes with 1-phenyl derivatives as in 1 reveal a square planar structure trans-[Ni(L(1)(-3)(*))(2)], whereas the corresponding 1,4-diphenyl derivatives are distorted tetrahedral as is demonstrated by X-ray crystallography of [Ni(L(5)(*))(2)] (5) and [Ni(L(6)(*))(2)] (6). Both series of mononuclear complexes possess a diamagnetic ground state. The electronic structures of both series have been elucidated experimentally (electronic spectra magnetization data). The square planar complexes 1-3 consist of a diamagnetic central Ni(II) ion and two strongly antiferromagnetically coupled ligand pi-radicals as has been deduced from correlated ab initio calculations; they are singlet diradicals. The tetrahedral complexes 4-6 consist of a paramagnetic high-spin Ni(II) ion (S(Ni) = 1), which is strongly antiferromagnetically coupled to two ligand pi-radicals. This is clearly revealed by DFT and correlated ab initio calculations. Electrochemically, complexes 1-6 can be reduced to form stable, paramagnetic monoanions [1-6](-) (S = (1)/(2)). The anions [1-3](-) are square planar Ni(II) (d,(8) S(Ni) = 0) species where the excess electron is delocalized over both ligands (class III, ligand mixed valency). In contrast, one-electron reduction of 4, 5, and 6 yields paramagnetic tetrahedral monoanions (S = (1)/(2)). X-band EPR spectroscopy shows that there are two different isomers A and B of each monoanion present in solution. In these anions, the excess electron is localized on one ligand [Ni(II)(L(4-6)(*))(L(4-6))](-) where (L(4-6))(2-) is the closed shell dianion of the ligands H(2)[L(4-6)] as was deduced from their electronic spectra and broken symmetry DFT calculations. Oxidation of 1 and 5 with excess iodine yields octahedral complexes [Ni(II)(L(1,ox))(2)I(2)] (7), [Ni(II)(L(1,ox))(3)](I(3))(2) (8), and trans-[Ni(II)(L(5,ox))(2)(I(3))(2)] (9), which have been characterized by X-ray crystallography; (L(1-)(6,ox)) represent the neutral, two-electron oxidized forms of the corresponding dianions (L(1-6))(2-). The room-temperature structures of complexes 1, 5, and 7 have been described previously in refs 1-5.     

Coenzyme B induced coordination of coenzyme M via its thiol group to Ni(I) of F430 in active methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the reaction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. At the active site, it contains the nickel porphinoid F430, which has to be in the Ni(I) oxidation state for the enzyme to be active. How the substrates interact with the active site Ni(I) has remained elusive. We report here that coenzyme M (HS-CoM), which is a reversible competitive inhibitor to methyl-coenzyme M, interacts with its thiol group with the Ni(I) and that for interaction the simultaneous presence of coenzyme B is required. The evidence is based on X-band continuous wave EPR and Q-band hyperfine sublevel correlation spectroscopy of MCR in the red2 state induced with 33S-labeled coenzyme M and unlabeled coenzyme B.     

Model studies of methyl CoM reductase: methane formation via CH3-S bond cleavage of Ni(I) tetraazacyclic complexes having intramolecular methyl sulfide pendants

The Ni(I) tetraazacycles [Ni(dmmtc)](+) and [Ni(mtc)](+), which have methylthioethyl pendants, were synthesized as models of the reduced state of the active site of methyl coenzyme M reductase (MCR), and their structures and redox properties were elucidated (dmmtc, 1,8-dimethyl-4,11-bis{(2-methylthio)ethyl}-1,4,8,11-tetraaza-1,4,8,11-cyclotetradecane; mtc, 1,8-{bis(2-methylthio)ethyl}-1,4,8,11-tetraaza-1,4,8,11-cyclotetradecane). The intramolecular CH(3)-S bond of the thioether pendant of [Ni(I)(dmmtc)](OTf) was cleaved in THF at 75 °C in the presence of the bulky thiol DmpSH, which acts as a proton source, and methane was formed in 31% yield and a Ni(II) thiolate complex was concomitantly obtained (Dmp = 2,6-dimesityphenyl). The CH(3)-S bond cleavage of [Ni(I)(mtc)](+) also proceeded similarly, but under milder conditions probably due to the lower potential of the [Ni(I)(mtc)](+) complex. These results indicate that the robust CH(3)-S bond can be homolytically cleaved by the Ni(I) center when they are properly arranged, which highlights the significance of the F430 Ni environment in the active site of the MCR protein.     

Structural, spectroscopic, and electrochemical studies of the complexes [Ni2(mu-CNR)(CNR)2(mu-dppm)2](n+) (n = 0, 1, 2): unusual examples of nickel(0)-nickel(I) and nickel(0)-nickel(II) mixed valency

Reaction of Ni(COD)(2) (COD = cyclooctadiene) with dppm (dppm = bis(diphenylphosphino) methane) followed by addition of alkyl or aryl isocyanides yields the class of nickel(0) dimers Ni(2)(mu-CNR)(CNR)(2)(mu-dppm)(2) (R = CH(3) (1), n-C(4)H(9) (2), CH(2)C(6)H(5) (3), i-C(3)H(7) (4), C(6)H(11) (5), t-C(4)H(9) (6), p-IC(6)H(4) (7), 2,6-(CH(3))(2)C(6)H(3) (8)). The cyclic voltammograms of the dimers exhibit two sequential single electron oxidations to the +1 and +2 forms. Specular reflectance infrared spectroelectrochemical (IRSEC) measurements demonstrate reversible interconversions between the neutral Ni(0) dimers and their +1 and +2 forms. Bulk samples of the +2 forms are prepared by chemical oxidation using [FeCp(2)][PF(6)], while the +1 forms are prepared by the comproportionation of neutral and +2 forms. The neutral complexes 6 and 8 were characterized by X-ray diffraction as symmetric, locally tetrahedral binuclear Ni(0) complexes. The +2 forms of these complexes, 6(2+) and 8(2+), have asymmetric structures with one locally square planar and one locally tetrahedral metal center, evidence for a Ni(II)-Ni(0) mixed valence state. The X-ray structural characterization of 6(+) is symmetrical and qualitatively similar to that of the neutral complex 6. The +1 forms all exhibit intense near IR electronic absorptions that are assigned as intervalence charge transfer (IVCT) bands. On the basis of structural, spectroscopic, and electrochemical data, the +1 forms of the complexes, 1(+)-8(+), are assigned as Robin-Day class III, fully delocalized Ni(+0.5)-Ni(+0.5) mixed valence complexes.     

Bis(alpha-diimine)nickel complexes: molecular and electronic structure of three members of the electron-transfer series [Ni(L)(2)](z)() (z = 0, 1+, 2+) (L = 2-Phenyl-1,4-bis(isopropyl)-1,4-diazabutadiene). A combined experimental and theoretical study

From the reaction of Ni(COD)(2) (COD = cyclooctadiene) in dry diethylether with 2 equiv of 2-phenyl-1,4-bis(isopropyl)-1,4-diazabutadiene (L(Ox))(0) under an Ar atmosphere, dark red, diamagnetic microcrystals of [Ni(II)(L*)(2)] (1) were obtained where (L*)(1-) represents the pi radical anion of neutral (L(Ox))(0) and (L(Red))(2-) is the closed shell, doubly reduced form of (L(Ox))(0). Oxidation of 1 with 1 equiv of ferrocenium hexafluorophosphate in CH(2)Cl(2) yields a paramagnetic (S = 1/2), dark violet precipitate of [Ni(I)(L(Ox))(2)](PF(6)) (2) which represents an oxidatively induced reduction of the central nickel ion. From the same reaction but with 2 equiv of [Fc](PF(6)) in CH(2)Cl(2), light green crystals of [Ni(II)(L(Ox))(2)(FPF(5))](PF(6)) (3) (S = 1) were obtained. If the same reaction was carried out in tetrahydrofuran, crystals of [Ni(II)(L(Ox))(2)(THF)(FPF(5))](PF(6)) x THF (4) (S = 1) were obtained. Compounds 1, 2, 3, and 4 were structurally characterized by X-ray crystallography: 1 and 2 contain a tetrahedral neutral complex and a tetrahedral monocation, respectively, whereas 3 contains the five-coordinate cation [Ni(II)(L(Ox))(2)(FPF(5))](+) with a weakly coordinated PF(6)(-) anion and in 4 the six-coordinate monocation [Ni(II)(L(Ox))(2)(THF)(FPF(5))](+) is present. The electro- and magnetochemistry of 1-4 has been investigated by cyclic voltammetry and SQUID measurements. UV-vis and EPR spectroscopic data for all compounds are reported. The experimental results have been confirmed by broken symmetry DFT calculations of [Ni(II)(L*)(2)](0), [Ni(I)(L(Ox))(2)](+), and [Ni(II)(L(Ox))(2)](2+) in comparison with calculations of the corresponding Zn complexes: [Zn(II)((t)L(Ox))(2)](2+), [Zn(II)((t)L(Ox))((t)L*)](+), [Zn(II)((t)L*)(2)](0), and [Zn(II)((t)L*)((t)L(Red))](-) where ((t)L(Ox))(0) represents the neutral ligand 1,4-di-tert-butyl-1,4-diaza-1,3-butadiene and ((t)L*)(1-) and ((t)L(Red))(2-) are the corresponding one- and two-electron reduced forms. It is clearly established that the electronic structures of both paramagnetic monocations [Ni(I)(L(Ox))(2)](+) (S = 1/2) and [Zn(II)((t)L(Ox))((t)(L*)](+) (S = 1/2) are different.     

Tetradentate bis(o-iminobenzosemiquinonate(1-)) pi radical ligands and their o-aminophenolate(1-) derivatives in complexes of nickel(II), palladium(II), and copper(II)

The coordination chemistries of the potential tetradentate ligands N,N'-bis(3,5-di-tert-butyl-2-hydroxyphenyl)ethylenediamine, H4[L1], the unsaturated analogue glyoxal-bis(2-hydroxy-3,5-di-tert-butylanil), H2[L2], and N,N'-bis(2-hydroxy-3,5-di-tert-butylphenyl)-2,2-dimethylpropylenediamine, H4[L3], have been investigated with nickel(II), palladium(II), and copper(II). The complexes prepared and characterized are [Ni(II)(H3L1)2] (1), [Ni(II)(HL2)2].5/8CH2Cl2 (2), [Ni(II)(L3**)] (3), [Pd(II)(L3**)][Pd(II)(H2L3) (4), and [Cu(II)(H2O)(L4)] (5), where (L4)2- is the oxidized diimine form of (L3)4- and (L3**)2- is the bis(o-iminosemiquinonate) diradical form of (L3)4-. The structures of compounds 1-5 have been determined by single crystal X-ray crystallography. In complexes 1 and 2, the ligands (H3L1)- and (HL2)- are tridentate and the nickel ions are in an octahedral ligand environment. The oxidation level of the ligands is that of an aromatic o-aminophenol. 1 and 2 are paramagnetic (mu(eff) approximately 3.2 mu(B) at 300 K), indicating an S = 1 ground state. The diamagnetic, square planar, four-coordinate complexes 3 and [Pd(II)(L3**)] in 4 each contain two antiferromagnetically coupled o-iminobenzosemiquinonate(1-) pi radicals. Diamagnetic [Pd(II)(H2L3)] in 4 forms an eclipsed dimer via four N-H.O hydrogen bonding contacts which yields a nonbonding Pd.Pd contact of 3.0846(4) A. Complex 5 contains a five-coordinate Cu(II) ion and two o-aminophenolate(1-) halves in (L4)2-. The electrochemistries of complexes 3 and 4a ([Pd(II)(L3**)] of 4) have been investigated, and the EPR spectra of the monocations and -anions are reported.     

Electronic structure of mononuclear bis(1,2-diaryl-1,2-ethylenedithiolato)iron complexes containing a fifth cyanide or phosphite ligand: a combined experimental and computational study

A series of mononuclear square-based pyramidal complexes of iron containing two 1,2-diaryl-ethylene-1,2-dithiolate ligands in various oxidation levels has been synthesized. The reaction of the dinuclear species [Fe(III)2(1L*)2(1L)2]0, where (1L)2- is the closed shell di-(4-tert-butylphenyl)-1,2-ethylenedithiolate dianion and (1L*)1- is its one-electron-oxidized pi-radical monoanion, with [N(n-Bu)4]CN in toluene yields dark green crystals of mononuclear [N(n-Bu)4][Fe(II)(1L*)2(CN)] (1). The oxidation of 1 with ferrocenium hexafluorophosphate yields blue [Fe(III)(1L*)2(CN)] (1ox), and analogously, a reduction with [Cp2Co] yields [Cp2Co][N(n-Bu)4][Fe(II)(1L*)(1L)(CN)] (1red); oxidation of the neutral dimer with iodine gives [Fe(III)(1L*)2I] (2). The dimer reacts with the phosphite P(OCH3)3 to yield [Fe(II)(1L*)2{P(OCH3)3}] (3), and [Fe(III)2(3L*)2(3L)2] reacts with P(OC6H5)3 to give [Fe(II)(3L*)2{P(OC6H5)3}] (4), where (3L)2- represents 1,2-diphenyl-1,2-ethylenedithiolate(2-). Both 3 and 4 were electrochemically one-electron oxidized to the monocations 3ox and 4ox and reduced to the monoanions 3red and 4red. The structures of 1 and 4 have been determined by X-ray crystallography. All compounds have been studied by magnetic susceptibility measurements, X-band EPR, UV-vis, IR, and M?ssbauer spectroscopies. The following five-coordinate chromophores have been identified: (a) [Fe(III)(L*)2X]n, X = CN-, I- (n = 0) (1ox, 2); X = P(OR)3 (n = 1+) )3ox, 4ox) with St = 1/2, SFe = 3/2; (b) [Fe(II)(L*)2X]n, X = CN-, (n = 1-) (1); X = P(OR)3 (n = 0) (3, 4) with St = SFe = 0; (c) [Fe(II)(L*)(L)X]n <--> [Fe(II)(L)(L*)X]n, X = CN- (n = 2-) (1red); X = P(OR)3 (n = 1-) (3red, 4red) with St = 1/2, SFe = 0 (or 1). Complex 1ox displays spin crossover behavior: St = 1/2 <--> St = 3/2 with intrinsic spin-state change SFe = 3/2 <--> SFe = 5/2. The electronic structures of 1 and 1(ox) have been established by density functional theoretical calculations: [Fe(II)(1L*)2(CN)]1- (SFe = 0, St = 0) and [Fe(III)(1L*)2(CN)]0 (SFe = 3/2, St = 1/2).     

Rapid ligand exchange in the MCRred1 form of methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis (Mtm), catalyses the final step in methane synthesis in all methanogenic organisms. Methane is produced by coenzyme B-dependent two-electron reduction of methyl-coenzyme M. At the active site of MCR is the corphin cofactor F(430), which provides four-coordination through the pyrrole nitrogens to a central Ni ion in all states of the enzyme. The important MCRox1 ("ready") and MCRred1 ("active") states contain six-coordinate Ni(I) and differ in their upper axial ligands; furthermore, red1 appears to be two-electrons more reduced than in ox1 and other Ni(II) states that have been studied. On the basis of the reactivity of MCRred1 and MCRox1 with a substrate analogue and inhibitor (3-bromopropanesulfonate) and other small molecules (chloroform, dichloromethane, mercaptoethanol, and nitric oxide), we present evidence that the six-coordinate Ni(I) centers in the MCRred1 and MCRox1 states exhibit markedly different inherent reactivities. MCRred1 reacts faster with chloroform (2100-fold or 35000-fold when corrected for temperature effects), nitric oxide (90-fold), and 3-bromopropanesulfonate (10(6)-fold) than MCRox1. MCRred1 reacts with chloroform and dichloromethane and, like F(430), can catalyze dehalogenation reactions and produce lower halogenated products. We conclude that the enhanced reactivity of MCRred1 is due to the replacement of a relatively exchange-inert thiol ligand in MCRox1 with a weakly coordinating upper axial ligand in red1 that can be easily replaced by incoming ligands.     

Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping‐Electron Paramagnetic Resonance Approach

Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read‐out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter‐copper electron transfer catalyzed by CuNiRs. Cryoreduction‐EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox‐center containing proteins (homo and hetero metal ions).     

Electrochemically induced chemically reversible proton-coupled electron transfer reactions of riboflavin (vitamin B2)

The electrochemical behavior of the naturally occurring vitamin B(2), riboflavin (Fl(ox)), was examined in detail in dimethyl sulfoxide solutions using variable scan rate cyclic voltammetry (ν = 0.1 - 20 V s(-1)) and has been found to undergo a series of proton-coupled electron transfer reactions. At a scan rate of 0.1 V s(-1), riboflavin is initially reduced by one electron to form the radical anion (Fl(rad)(?-)) at E(0)(f) = -1.22 V versus Fc/Fc(+) (E(0)(f) = formal reduction potential and Fc = ferrocene). Fl(rad)(?-) undergoes a homogeneous proton transfer reaction with the starting material (Fl(ox)) to produce Fl(rad)H(?) and Fl(ox)(-), which are both able to undergo further reduction at the electrode surface to form Fl(red)H(-) (E(0)(f) = -1.05 V vs Fc/Fc(+)) and Fl(rad)(?2-) (E(0)(f) = -1.62 V vs Fc/Fc(+)), respectively. At faster voltammetric scan rates, the homogeneous reaction between Fl(rad)(?-) and Fl(ox) begins to be outrun, which leads to the detection of a voltammetric peak at more negative potentials associated with the one-electron reduction of Fl(rad)(?-) to form Fl(red)(2-) (E(0)(f) = -1.98 V vs Fc/Fc(+)). The variable scan rate voltammetric data were modeled quantitatively using digital simulation techniques based on an interconnecting "scheme of squares" mechanism, which enabled the four formal potentials as well as the equilibrium and rate constants associated with four homogeneous reactions to be determined. Extended time-scale controlled potential electrolysis (t > hours) and spectroscopic (EPR and in situ UV-vis) experiments confirmed that the chemical reactions were completely chemically reversible.     

Autoredox interconversion of two galactose oxidase forms GOase(ox) and GOase(semi) with and without dioxygen

Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOase(ox)(Cu(II)-Tyr(*)) and GOase(semi)(Cu(II)-Tyr), as a result of processes involving the formation and decay of the Cu(II)-coordinated tyrosyl radical (Tyr(*)). In this work the two reactions have been studied by UV-vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOase(ox) to GOase(semi), which in air-free conditions is 100% complete. Rate constants (k(red)) are dependent on pH, and previously defined acid dissociation constants pK(1a) = 5.7 (exogenous H(2)O ligand), and pK(2a) = 8.0 (axial H(+)Tyr-495) apply. Values of k(red)(25 degrees C) range from 1.55 x 10(-4) s(-1) (pH 5.5) to 2.69 x 10(-4) s(-1) (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N(3)(-) or NCS(-) present in amounts sufficient to give >98% binding at the substrate binding (exogenous) site, while CH(3)CO(2)(-) and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 degrees C) is 161 M(-1) at pH 6.4 for acetate (previous value 140 M(-)(1)) and 46 M(-1) at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 A from the Cu) is chemically modified with HSPO(3)(2-), and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOase(semi) to GOase(ox) only occurs with O(2) present, when a first-order dependence on [O(2)] is observed, giving k(ox)(25 degrees C) = 0.021 M(-1) s(-1) at pH 7.5. This process is unaffected by NCS(-) or N(3)(-) bound at the exogenous site, and a mechanism involving outer-sphere reaction of O(2) to O(2)(-) followed by a fast step O(2) to H(2)O(2) is proposed. As GOase(ox) is formed, autoreduction back to GOase(semi) occurs, and at pH 7.5 with O(2) in large excess (1.13 mM) the maximum conversion to GOase(ox) is 69%. The k(ox) reaction proceeds to completion with >98% N(3)(-) bound at the exogenous site.     

Stopped-Flow Kinetics of Methyl Group Transfer between the Corrinoid-Iron-Sulfur Protein and Acetyl-Coenzyme A Synthase from Clostridium thermoaceticum

Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the stopped-flow method at 390 nm. Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) <==> Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations. Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)). The simplest mechanism that adequately fit the data involved formation of a [CH(3)-Co(3+)FeSP]:[ACS(red)] complex, methyl group transfer (forming [Co(1+)FeSP]:[CH(3)-ACS(ox)]), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO <==> ACS(red)-CO). Best-fit rate constants were obtained. CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM. Fits were unimproved when >1 CO was assumed to bind. Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox). This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction. Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively. Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)). The nucleophilicity of the [Ni(2+)D(red)] unit appears comparable to that of Co(1+) cobalamins. Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex.     

Redox-active p-quinone-based Bis(pyrazol-1-yl)methane ligands: synthesis and coordination behaviour

The synthesis, structural characterisation and coordination behaviour of mono- and ditopic p-hydroquinone-based bis(pyrazol-1-yl)methane ligands is described (i.e., 2-(pz2CH)C6H3(OH)2 (2a), 2-(pz2CH)-6-(tBu)C6H2(OH)2 (2b), 2-(pz2CH)-6-(tBu)C6H2(OSiiPr3)(OH) (2c), 2,5-(pz2CH)2C6H2(OH)2 (4)). Ligands 2a, 2b and 4 can be oxidised to their p-benzoquinone state on a preparative scale (2a ox, 2b ox, 4 ox). An octahedral Ni II complex [trans-Ni(2c)2] and square-planar Pd II complexes [Pd2bCl2] and [Pd2b ox Cl2] have been prepared. In the two Pd II species, the ligands are coordinated only through their pyrazolyl rings. The fact that [Pd2bC12] and [Pd2b oxC12] are isolable compounds proves that redox transitions involving the p-quinone substituent are fully reversible. In [Pd2b oxCl2], the methine proton is highly acidic and can be abstracted with bases as weak as NEt(3). The resulting anion dimerises to give a dinuclear macrocyclic Pd II complex, which has been structurally characterised. The methylated ligand 2-(pz2CMe)C6H3O2 (11 ox) and its Pd II complex [Pd11 oxCl2] are base-stable. A new class of redox-active ligands is now available with the potential for applications both in catalysis and in materials science.     

Structure and properties of bivalent nickel and copper complexes with pyrazine-amide-thioether coordination: stabilization of trivalent nickel

Acyclic pyrazine-2-carboxamide and thioether containing hexadentate ligand 1,4-bis[o-(pyrazine-2-carboxamidophenyl)]-1,4-dithiobutane (H(2)bpzctb), in its deprotonated form, has afforded light brown [Ni(II)(bpzctb)](1)(S=1) and green [Cu(II)(bpzctb)](2)(S=1/2) complexes. The crystal structures of 1.CH(3)OH and 2.CH(2)Cl(2) revealed that in these complexes the ligand coordinates in a hexadentate mode, affording examples of distorted octahedral M(II)N(2)(pyrazine)N'(2)(amide)S(2)(thioether) coordination. Each complex exhibits in CH(2)Cl(2) a reversible to quasireversible cyclic voltammetric response, corresponding to the Ni(III)/Ni(II)(1) and Cu(II)/Cu(I)(2) redox process. The E(1/2) values reveal that the complexes of bpzctb(2-) are uniformly more anodic by approximately 0.2 V than those of the corresponding complexes with the analogous pyridine ligand, 1,4-bis[o-(pyridine-2-carboxamidophenyl)]-1,4-dithiobutane (H(2)bpctb), attesting that compared to pyridine, pyrazine is a better stabilizer of the Ni(ii) or Cu(i) state. Coulometric oxidation of the previously reported complex [Ni(II)(bpctb)] and 1 generates [Ni(III)(bpctb)](+) and [Ni(III)(bpzctb)](+) species, which exhibit a LMCT transition in the 470--480 nm region and axial EPR spectra corresponding to a tetragonally elongated octahedral geometry. Complex 2 exhibits EPR spectra characteristic of the d(z(2)) ground state.