Question 1: The FeS/H2S System as a Possible Primordial Source of Redox Energy

The theory of chemoautotrophy, as developed by Wächtershäuser, has been subject to experimental studies, which show a possible carbon fixation pathway of several consecutive steps from simple CO₂ to amino acids, using the redox system of iron sulphide and hydrogen sulphide. Main findings were a mimicking of the acetyl-CoA enzyme reaction using the mixed sulphide (Fe,Ni)S and the reduction of dinitrogen to ammonia. Present studies aim at a more detailed investigation of the mechanism of the redox system FeS/H₂S and its properties. For these studies a method to produce and immobilise FeS nanoparticles has been developed.     

Enhanced efficiency of ATP hydrolysis during nitrogenase catalysis utilizing reductants that form the all-ferrous redox state of the Fe protein

The amount of MgATP hydrolyzed per pair of electrons transferred (ATP/2e) during nitrogenase catalysis (1.0 atm N(2), 30 degrees C) using titanium(III) citrate (Ti(III)) as reductant was measured and compared to the same reaction using dithionite (DT). ATP/2e values near 2.0 for Ti(III) and 5.0 for DT indicate that nitrogenase has a much lower ATP requirement using Ti(III) as reductant. Using reduced Azotobacter vinelandii flavoprotein (AvFlpH(2)), a possible in vivo nitrogenase reductant, ATP/2e values near 2.0 were also observed. When the reaction was conducted using Ti(III) under N(2), 5% CO in N(2), Ar, 5% CO in Ar, or acetylene, ATP/2e values near 2.0 were also observed. With Ti(III) as reductant, ATP/2e values near 2.0 were measured as a function of temperature, Fe:MoFe protein ratio, and MoFe:Fe protein ratio, in contrast to measured values of 4.0-25 when DT is used under the same conditions. Both Ti(III) and AvFlpH(2) are capable of forming the [Fe(4)S(4)](0) cluster state of the Fe protein whereas DT is not, suggesting that ATP/2e values near 2.0 arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with hydrolysis of only 2 ATPs per pair of electrons transferred. Additional experiments showed that ATP/2e values near 2. 0 correlated with slower rates of product formation and that faster rates of product formation produced ATP/2e values near 5.0. ATP/2e values of 5.0 are consistent with the operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple while ATP/2e values of 2.0 could arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple. These results suggest that two distinct Fe protein redox couples may be functional during nitrogenase catalysis and that the efficiency of ATP utilization depends on which of these redox couples is dominant.     

Redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Redox titrations of carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum were performed using the reductant CO and the oxidant thionin. Titrations were followed at 420 nm, a wavelength sensitive to redox changes of the iron-sulfur clusters in the enzyme. When CODH was oxidized by just enough thionin to maximize A420, two molecules of CO per mole of CODH dimer (4 equiv/mol) reduced the enzyme fully. Likewise, 4 equiv/mol of thionin oxidized the fully-reduced enzyme to the point where A420 maximized. The four n = 1 redox sites which titrated in this region were designated group I sites. They include at least two iron-sulfur clusters, [Fe/S]A and [Fe/S]B, and two other sites, A' and B'. The [Fe4S4]2+/1+ cluster in CODH is included in this group. [Fe/S]B and B' have reduction potentials (at pH 8) below -480 mV vs NHE; [Fe/S]A and A' have reduction potentials above that value. The reduction potential of either [Fe/S]B or B' is near to the CO/CO2 couple at pH 8 (-622 mV). When CODH was oxidized by more than enough thionin to maximize A420, some of the excess thionin oxidized the so-called group II redox sites. These sites have reduction potentials more positive than group I and do not exhibit changes at 420 nm when titrated. Titration of group II sites required 1-2 equiv/mol. EPR of reduced group II sites exhibited the gav = 1.82 signal. When these sites were oxidized, the only signal present had g values at 2.075, 2.036, and 1.983.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of N2 by Fe2+ via Homogeneous and Heterogeneous Reactions

Because of their high kinetic barrier and thermodynamic favorability under moderately reducing atmospheric composition photochemical approaches appear to be ideally suited to the direct reduction of solvated nitrogen gas. Ferrous iron has been investigated as an electron donor, as it has a highly tunable redox character and is environmentally ubiquitous. Recent advances in mineralogy connected to the field of environmental remediation have led to the identification of an important class of rusts which have reductive potentials comparable to Fe(0). These materials possess redox couples that are, potentially, capable of overcoming the kinetic barrier to the production of NH₃. In this study, we attempted to produce ammonia from N₂ by oxidizing white rust both photochemically and in a dark reaction. All results indicated the reaction was inhibited by competing reactions; primarily the reduction of H₂O to H₂. However, the dark reactions showed limited potential for reduction up to 1.4 mM. As a result, we turned to the question of closure temperature; the minimum temperature of rapid reaction based on a choice of reductant, which we demonstrate a model for its estimation. Due to the high thermodynamic energy of the intermediate, we conclude that aqueous photochemical reduction under the conditions studied here is an unlikely prebiotic source for reactive, i.e. reduced, nitrogen.     

Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme: a pulse radiolysis study

We have studied the one-electron reduction of oxidized Chlamydomonas reinhardtii thioredoxin and compared it to that of hen egg white lysozyme, using CO(2)(*) (-) free radicals as reductants. This comparison shows that the thioredoxin disulfide/thiol redox couple has different properties than that of lysozyme: the disulfide radical pK(a) is much lower (around 5 for small disulfides, 4.62 for lysozyme, <3 for thioredoxin). To get a better understanding of the modulation of the thioredoxin redox properties we have constructed the mutants W35A and D30A. Their reduction by pulse radiolysis indicates that W35 strongly controls both the disulfide radical acidity (the pK(a) in W35A is equal to ca. 4), and the thiol reactivity. Asp30 is also involved in the control of proton transfer to the disulfide free radical. In addition, its removal seems to increase the reduction potential of the thioredoxin thiyl/thiol couple. Overall, the reduction properties of thioredoxin confirm its nature as a unique reductant.     

CO dehydrogenase from Clostridium thermoaceticum. EPR and electrochemical studies in CO2 and argon atmospheres

The EPR and redox properties of the metal complexes in CO dehydrogenase (CODH) from Clostridium thermoaceticum were studied. Controlled potential coulometric reductive titrations of CODH were performed under argon and CO2 atmospheres. In the titrations performed under argon, five to eight electrons/dimer were required for reduction, and four distinct EPR signals appeared. These included a signal with gave = 1.82 (Em approximately -220 mV), two signals with the same g values but different linewidths at gave = 1.94 (Em approximately -440 mV), and a signal at gave = 1.86 (Em approximately -530 mV). All of the S = 1/2 EPR signals had low spin concentrations; values between 0.2 and 0.3 spins/dimer were typically obtained for each signal. Features between g = 6 and 4, typical of S = 3/2 states, were also observed, and these may account, at least to some degree, for the low spin concentration values. Under CO2, and at negative potentials, CODH served as an electrocatalyst in the reduction of CO2 to CO. The apparent half-maximal activity for this reduction at pH 6.3 occurred at -430 mV, a potential near the thermodynamic value. An EPR signal, arising from a complex containing Ni, Fe, and the carbon from CO/CO2 developed along with this activity. The reduction of this complex is probably the last step to occur prior to the catalysis of CO2 reduction.     

Photoelectrochemical Power,Chemical Energy and Catalytic Activity for Organic Evolution on Natural Pyrite Interfaces

Natural pyrite (FeS₂) has frequently been discussed as a material involved in CO₂ fixation in presence of H₂S and as a possible catalyst for the origin of life. A straightforward chemical fixation of carbon dioxide as proposed by Wächtershäuser could not be verified from thermo-chemical equilibrium calculations by minimizing Gibb's Free Energy in the system C, O, H, S, Fe and appears unlikely dueto the experimentally encountered large overpotentials involved in CO₂ fixation. However, the hypothesis, by W. R. Edwards, that pyrite in shallow coastal waters may have been involved, canbe sustained. In this case, daily available photoelectrochemical power from FeS₂/Fe^2+/3+ interfaces could have made thedifference in combination with electrochemical processes, such ashydrogen insertion, and the solubilization of pyrite by the aminoacid cysteine to yield dissolved chemical energy. Periodical changes in energy supply could also have entrained primitive self-organization processes for organic-biological evolution.Natural samples from thirteen ore deposits have been investigatedphotoelectrochemically. Efficient light-induced current generation has been found with several of these samples so thatphotoelectrochemical processes generated by pyrite have to be considered as naturally occurring phenomena, which could have been even more pronounced in oxygen deficient environments. Pyrite from the Murgul mine in Turkey of suboceanic volcanicorigin was closer examined as a model system to understand the morphology and chemistry of pyrite photoactivity.     

Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and bacteria

Thermophilic and hyperthermophilic Archaea and Bacteria have been isolated from marine hydrothermal systems, heated sediments, continental solfataras, hot springs, water heaters, and industrial waste. They catalyze a tremendous array of widely varying metabolic processes. As determined in the laboratory, electron donors in thermophilic and hyperthermophilic microbial redox reactions include H2, Fe(2+), H2S, S, S2O3(2-), S4O6(2-), sulfide minerals, CH4, various mono-, di-, and hydroxy-carboxylic acids, alcohols, amino acids, and complex organic substrates; electron acceptors include O2, Fe(3+), CO2, CO, NO3(-), NO2(-), NO, N2O, SO4(2-), SO3(2-), S2O3(2-), and S. Although many assimilatory and dissimilatory metabolic reactions have been identified for these groups of microorganisms, little attention has been paid to the energetics of these reactions. In this review, standard molal Gibbs free energies (DeltaGr(0)) as a function of temperature to 200 degrees C are tabulated for 370 organic and inorganic redox, disproportionation, dissociation, hydrolysis, and solubility reactions directly or indirectly involved in microbial metabolism. To calculate values of DeltaGr(0) for these and countless other reactions, the apparent standard molal Gibbs free energies of formation (DeltaG(0)) at temperatures to 200 degrees C are given for 307 solids, liquids, gases, and aqueous solutes. It is shown that values of DeltaGr(0) for many microbially mediated reactions are highly temperature dependent, and that adopting values determined at 25 degrees C for systems at elevated temperatures introduces significant and unnecessary errors. The metabolic processes considered here involve compounds that belong to the following chemical systems: H-O, H-O-N, H-O-S, H-O-N-S, H-O-C(inorganic), H-O-C, H-O-N-C, H-O-S-C, H-O-N-S-C(amino acids), H-O-S-C-metals/minerals, and H-O-P. For four metabolic reactions of particular interest in thermophily and hyperthermophily (knallgas reaction, anaerobic sulfur and nitrate reduction, and autotrophic methanogenesis), values of the overall Gibbs free energy (DeltaGr) as a function of temperature are calculated for a wide range of chemical compositions likely to be present in near-surface and deep hydrothermal and geothermal systems.     

Evidence for a two-electron transfer using the all-ferrous Fe protein during nitrogenase catalysis

The nitrogenase-catalyzed H(2) evolution and acetylene-reduction reactions using Ti(III) and dithionite (DT) as reductants were examined and compared under a variety of conditions. Ti(III) is known to make the all-ferrous Fe protein ([Fe(4)S(4)](0)) and lowers the amount of ATP hydrolyzed during nitrogenase catalysis by approximately 2-fold. Here we further investigate this behavior and present results consistent with the Fe protein in the [Fe(4)S(4)](0) redox state transferring two electrons ([Fe(4)S(4)](2+)/[Fe(4)S(4)](0)) per MoFe protein interaction using Ti(III) but transferring only one electron ([Fe(4)S(4)](2+)/[Fe(4)S(4)](1+)) using DT. MoFe protein specific activity was measured as a function of Fe:MoFe protein ratio for both a one- and a two-electron transfer reaction, and nearly identical curves were obtained. However, Fe protein specific activity curves as a function of MoFe:Fe protein ratio showed two distinct reactivity patterns. With DT as reductant, typical MoFe inhibition curves were obtained for operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple, but with Ti(III) as reductant the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple was functional and MoFe inhibition was not observed at high MoFe:Fe protein ratios. With Ti(III) as reductant, nitrogenase catalysis produced hyperbolic curves, yielding a V(max) for the Fe protein specific activity of about 3200 nmol of H(2) min(-1) mg(-1) Fe protein, significantly higher than for reactions conducted with DT as reductant. Lag phase experiments (Hageman, R. V., and Burris, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2699-2702) were carried out at MoFe:Fe protein ratios of 100 and 300 using both DT and Ti(III). A lag phase was observed for DT but, with Ti(III) product formation, began immediately and remained linear for over 30 min. Activity measurements using Av-Cp heterologous crosses were examined using both DT and Ti(III) as reductants to compare the reactivity of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) and [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couples and both were inactive. The results are discussed in terms of the Fe protein transferring two electrons per MoFe protein encounter using the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with Ti(III) as reductant.     

Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.     

Unusual low reactivity of the water oxidase in redox state S3 toward exogenous reductants. Analysis of the NH2OH- and NH2NH2-induced modifications of flash-induced oxygen evolution in isolated spinach thylakoids

The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].     

The reactivity of hydrazine with photosystem II strongly depends on the redox state of the water oxidizing system

The decay kinetics of the redox states S2 and S3 of the water-oxidizing enzyme have been analyzed in isolated spinach thylakoids in the absence and presence of the exogenous reductant hydrazine. In control samples without NH2NH2 a biphasic decay is observed. The rapid decline of S2 and S3 with YD as reductant exhibits practically the same kinetics with t1/2 = 6-7 s at pH = 7.2 and 7 degrees C. The slow reduction (order of 5-10 min at 7 degrees C) of S2 and S3 with endogenous electron donors other than YD is about twice as fast for S2 as for S3 under these conditions. In contrast, the hydrazine-induced reductive shifts of the formal redox states Si (i = 0...3) are characterized by a totally different kinetic pattern: (a) at 1 mM NH2NH2 and incubation on ice the decay of S2 is estimated to be at least 25 times faster (t1/2 less than or equal to 0.4 min) than the corresponding reaction of S3 (t1/2 approximately 13 min); (b) the NH2NH2-induced decay of S3 is even slower (about twice) than the transformation of S1 into the formal redox state 'S-1' (t1/2 approximately 6 min), which gives rise to the two-digit phase shift of the oxygen-yield pattern induced by a flash train in dark adapted thylakoids. (c) the NH2NH2-induced transformation S0----'S-2' [Renger, Messinger and Hanssum (1990) in: Curr.' Res. Photosynth. (Baltscheffsky, M., ed), Vol. 1, pp. 845-848, Kluwer, Dordrecht] is about three times faster (t1/2 approximately 2 min) than the reaction [see text]. Based on these results, the following dependence on the redox state Si of the reactivity towards NH2NH2 is obtained: S3 less than S1 less than S0 much less than S2. The implications of this surprising order of reactivity are discussed.     

ATR-FTIR redox difference spectroscopy of Yarrowia lipolytica and bovine complex I

ATR-FTIR spectroscopy in combination with electrochemistry has been applied to the redox centers of Yarrowia lipolytica complex I. The redox spectra show broad similarities with previously published data on Escherichia coli complex I and with new data here on bovine complex I. The spectra are dominated by amide I/II protein backbone changes. Comparisons with redox IR spectra of small model ferredoxins demonstrate that these amide I/II changes arise primarily from characteristic structural changes local to the iron-sulfur centers, rather than from global structural alterations as has been suggested previously. Bands arising from the substrate ubiquinone were evident, as was a characteristic 1405 cm(-)(1) band of the reduced form of the FMN cofactor. Other signals are likely to arise from perturbations or protonation changes of a carboxylic amino acid, histidine, and possibly several other specific amino acids. Redox difference spectra of center N2, together with substrate ubiquinone, were isolated from those of the other iron-sulfur centers by selective redox potentiometry. Its redox-linked amide I/II changes were typical of those in other 4Fe-4S iron sulfur proteins. Contrary to published data on bacterial complex I, no center N2 redox-linked protonation changes of carboxylic amino acids or tyrosine were evident, and other residues that could provide its redox-linked protonation site are discussed. Features of the substrate ubiquinone associated with the center N2 spectrum were particularly clear, with firm assignments possible for bands from both oxidized and reduced forms. This is the first report of IR properties of ubiquinone in complex I, and the data could be used to estimate a stoichiometry of 0.2-0.4 per complex I.     

Iron-based redox centres of reductase and oxygenase components of phenol hydroxylase from A. radioresistens: a redox chain working at highly positive redox potentials

This is the first report of the direct electrochemistry of the reductase (PHR) and oxygenase (PHO) components of phenol hydroxylase from Acinetobacter radioresistens S13 studied by cyclic and differential pulse voltammetry. The PHR contains one 2Fe2S cluster and one FAD that mediate the transfer of electrons from NAD(P)H to the non-heme diiron cluster of PHO. Cyclic and differential pulse voltammetry (CV and DPV) on glassy carbon showed two redox pairs with midpoint potentials at +131.5 ± 13 mV and -234 ± 3 mV versus normal hydrogen electrode (NHE). The first redox couple is attributed to the FeS centre, while the second one corresponds to free FAD released by the protein. DPV scans on native and guanidinium chloride treated PHR highlighted the presence of a split signal (ΔE ≈ 100 mV) attributed to heterogeneous properties of the 2Fe2S cluster interacting with the electrode, possibly due to the presence of two protein conformers and consistently with the large peak-to-peak separation and the peak broadening observed in CV. DPV experiments on gold electrodes performed on PHO confirm a consistently higher reduction potential at +396 mV vs. NHE. The positive redox potentials measured by direct electrochemistry for the FeS cluster in PHR and for the non-heme diiron cluster of PHO show that the entire phenol hydroxylase system works at higher potentials than those reported for structurally similar enzymes, for example methane monooxygenases.     

Reductive activation of the coenzyme A/acetyl-CoA isotopic exchange reaction catalyzed by carbon monoxide dehydrogenase from Clostridium thermoaceticum and its inhibition by nitrous oxide and carbon monoxide

The final steps in the synthesis of acetyl-CoA by CO dehydrogenase (CODH) have been studied by following the exchange reaction between CoA and the CoA moiety of acetyl-CoA. This reaction had been studied earlier (Pezacka, E., and Wood, H. G. (1986) J. Biol. Chem. 261, 1609-1615 and Ramer, W. E., Raybuck, S. A., Orme-Johnson, W. H., and Walsh, C. T. (1989) Biochemistry 28, 4675-4680). The CoA/acetyl-CoA exchange activity was determined at various controlled redox potentials and was found to be activated by a one-electron reduction with half-maximum activity occurring at -486 mV. There is approximately 2000-fold stimulation of the exchange by performing the reaction at -575 mV relative to the rate at -80 mV. Binding of CoA to CODH is not sensitive to the redox potential; therefore, the reductive activation affects some step other than association/dissociation of CoA. We propose that a metal center on CODH with a midpoint reduction potential of less than or equal to -486 mV is activated by a one-electron reduction to cleave the carbonyl-sulfur bond and/or bind the acetyl group of acetyl-CoA. Based on a comparison of the redox dependence of this reaction with that for methylation of CODH (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) and CO2 reduction and formation of the Ni-Fe-C EPR signal (Lindahl, P. A., Münck, E., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3873-3879), we propose that the assembly of the acetyl group of acetyl-CoA, i.e. binding the methyl group of the methylated corrinoid/iron-sulfur protein, binding CO, and methyl migration to form the acetyl-CODH intermediate, occur at the novel Ni-Fe3-4-containing site in CODH. CO has two effects on the CoA/acetyl-CoA exchange: it activates the reaction due to its reductive capacity and its acts as a noncompetitive inhibitor. We also discovered that the CoA/acetyl-CoA exchange was inhibited by nitrous oxide via an oxidative mechanism. In the presence of a low-potential electron donor, CODH becomes a nitrous oxide reductase which catalytically converts N2O to N2. This study combined with earlier results (Lu, W-P., Harder, S. R., and Ragsdale, S. W. (1990) J. Biol. Chem. 265, 3124-3133) establishes that the two-subunit form of CODH is completely active in all reactions known to be catalyzed by CODH.     

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of ?483 ± 1 mV and ?187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.     

[FeS/FeS2]. A redox system for the origin of life

The FeS/FeS₂ redox system, whose importance is stressed in recent theories on the origin of life, has been tested experimentally. In this paper it is demonstrated by thermodynamical calculations as well as by experiments, that cyclohexanone, which served as model compound, can be reduced by the aforementioned redox system. Reactions were carried out in methanol and DMF at 25 °C and at 100 °C. Besides products that were synthesised in both solvents, like cyclohexanethiol and dicyclohexyldisulphide, special compounds were obtained in methanol and in DMF, because of the involvement of the respective solvent in the reaction. Yields of reduced compounds were lower in methanol owing to a compound that hindered the reduction (cyclohexylketal). With increasing temperature and duration the amount of reduced compoinds increased. Further experiments have shown that 1,1-cyclohexanedithiol is likely to be a necessary intermediate for the reduced products. The experiments give evidence to the pyrite hypothesis, which postulates that the FeS/FeS₂ redox system was of importance for the origin of life.     

The Effects of Borate Minerals on the Synthesis of Nucleic Acid Bases,Amino Acids and Biogenic Carboxylic Acids from Formamide

The thermal condensation of formamide in the presence of mineral borates is reported. The products afforded are precursors of nucleic acids, amino acids derivatives and carboxylic acids. The efficiency and the selectivity of the reaction was studied in relation to the elemental composition of the 18 minerals analyzed. The possibility of synthesizing at the same time building blocks of both genetic and metabolic apparatuses, along with the production of amino acids, highlights the interest of the formamide/borate system in prebiotic chemistry.     

Evidence that ferredoxin interfaces with an internal redox shuttle in Acetyl-CoA synthase during reductive activation and catalysis

Acetyl-CoA synthase (ACS), a subunit of the bifunctional CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex of Moorella thermoacetica requires reductive activation in order to catalyze acetyl-CoA synthesis and related partial reactions, including the CO/[1-(14)C]-acetyl-CoA exchange reaction. We show that the M. thermoacetica ferredoxin(II) (Fd-II), which harbors two [4Fe-4S] clusters and is an electron acceptor for CODH, serves as a redox activator of ACS. The level of activation depends on the oxidation states of both ACS and Fd-II, which strongly suggests that Fd-II acts as a reducing agent. By the use of controlled potential enzymology, the midpoint reduction potential for the catalytic one-electron redox-active species in the CO/acetyl-CoA exchange reaction is -511 mV, which is similar to the midpoint reduction potential that was earlier measured for other reactions involving ACS. Incubation of ACS with Fd-II and CO leads to the formation of the NiFeC species, which also supports the role of Fd-II as a reductant for ACS. In addition to being a reductant, Fd-II can accept electrons from acetylated ACS, as observed by the increased intensity of the EPR spectrum of reduced Fd-II, indicating that there is a stored electron within an "electron shuttle" in the acetyl-Ni(II) form of ACS. This "shuttle" is proposed to serve as a redox mediator during activation and at different steps of the ACS catalytic cycle.     

Redox-dependent CO2 reduction activity of CO dehydrogenase from Rhodospirillum rubrum.

Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes both the oxidation of CO and the reduction of CO(2). Studies of the redox dependence of CO(2) reduction by R. rubrum CODH show that (1) CODH is unable to catalyze CO(2) reduction at potentials greater than -300 mV; (2) the maximum activity is observed at potentials less than -480 mV; and (3) the midpoint potential (E(m)) of the transition from minimum to maximum CO(2) reduction activity occurs at approximately -339 mV. These results indicate that the C(red1) state of R. rubrum CODH (E(m) = -110 mV; g(zyx)() = 2.03, 1.88, 1.71) is not competent to reduce CO(2). Nernst analyses suggest that the reduction of CODH from the C(red1) state to the CO(2)-reducing form (C(unc), g(zyx)() = 2.04, 1.93, 1.89; E < approximately -300 mV) of the enzyme is a one-electron process. For the entire redox range, viologens stimulate CO(2) reduction by CODH more than 50-fold, and it is proposed that viologens accelerate the redox equilibration of redox buffers and [Fe(4)S(4)](B) during catalysis.