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PubMed1995

Electron transfer reactions between aromatic amine dehydrogenase and azurin.

Binding and electron transfer reactions between the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady-state kinetic assays using azurin as an electron acceptor, it was observed that the apparent Km for azurin decreased with increasing ionic strength. These results are the opposite of what was observed for the reaction between the TTQ enzyme methylamine dehydrogenase (MADH) and amicyanin, despite the fact that in both cases the pairs of redox proteins are each acidic proteins. It was further demonstrated that azurin does not function as an effective electron acceptor for MADH, and that amicyanin does not function as an effective electron acceptor for AADH. Thus, while the two TTQ enzymes each use type I copper proteins as physiologic electron acceptors, there is a strong specificity for which copper protein serves as a redox partner. The kinetic parameters for the electron transfer reactions from reduced AADH to oxidized azurin were determined by stopped-flow spectroscopy. Different results were obtained depending upon whether AADH was reduced chemically with dithionite or with the substrate tyramine. The values for the limiting first-order apparent electron transfer rate constant (kET) at 30 degrees C were 4 and 102 s-1, respectively. Kinetically determined values of Kd also differed by a factor of 2.4. These data suggest that the incorporation of the substrate-derived amino group into the reduced TTQ of AADH significantly increases the apparent kET. The interaction between AADH and azurin was also quantitated using an ultrafiltration binding assay. This yielded a Kd of 300 microM for the AADH--azurin complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcaligenes↗
PubMed1993

Phototrapping of doubly reduced monomeric bacteriochlorophyll in the photoreaction center of Ectothiorhodospira sp.

The photoreaction center from the purple sulfur bacterium Ectothiorhodospira sp. was illuminated in the presence of reduced cytochrome c or dithionite under anaerobic conditions. This treatment first caused the monoelectronic reduction of both molecules of bacteriopheophytin (Bph), phi A and phi B, as witnessed by the appearance of EPR and optical signals typical of singly-reduced bacteriochlorins. Continued illumination under the same reducing conditions caused both of these signals to disappear. Such disappearance was accompanied by a complete bleaching of the Qx and Qy absorption bands of Bph but not of the corresponding transitions of bacteriochlorophyll (Bchl). These phenomena are interpreted by a double reduction of phi A and phi B. As long as the medium remained reducing and anaerobic, these changes were stable. Prolonged illumination under the same reducing conditions finally led to the bleaching of the Qx (600 nm) and Qy (800 nm) bands of Bchl but not of the 880-nm band. This generated no EPR or 645-nm absorption signals due to singly-reduced Bchl. The bleaching kinetics of the 800-nm band was biphasic and paralleled a shift of the peak wavelength. This is interpreted by a double reduction of both molecules of monomeric Bchl BA and BB in an undetermined order. After bleaching of the 800-nm band has reached saturation, the absorbance ratio of the 800/880-nm absorption bands remains constant, as would be expected if the ultimate spectrum was that of the primary electron donor. These experiments demonstrate the photoreduction of Bchl and allow the absorption spectrum of the primary donor to be measured for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗
PubMed1993

Role of phosphate in Fe2+ binding to horse spleen holoferritin.

In order to identify the function and location of phosphate associated with the iron core of horse spleen ferritin (HoSF), the phosphate content of native HoSF was altered by two procedures. Adjustment of pH from 7.0 to 10.0 irreversibly released 53% of the phosphate and 10% of the iron, while lowering the pH to 5.0 reversibly released 43% of the phosphate and 35% of the iron. Reversible release of 85% of the initial phosphate (but little iron release) also occurs upon reduction with methyl viologen (MV) or dithionite. Most of the phosphate is released in the early stages of reduction of the iron core, suggesting that the phosphate resides primarily on the mineral core surface. Reduction followed by chelation altered both the iron and phosphate content of the HoSF mineral cores. HoSF iron cores first reconstituted in the absence of phosphate and then incubated with added phosphate did not bind phosphate. However, when HoSF was first reconstituted in the absence of phosphate and then equilibrated anaerobically with both Fe2+ and phosphate, then phosphate was incorporated in amounts similar to native HoSF. Fe2+ binding to native, phosphate altered, and reconstituted HoSF in the presence and absence of phosphate clearly showed that Fe2+ binding to the mineral core depends on the presence of core-bound phosphate. Fe2+ binding to phosphate-depleted mineral cores or to cores reconstituted with 621, 2158, and 3013 Fe/HoSF core in the absence of phosphate bound only eight Fe2+ per entire ferritin molecule, clearly showing that Fe2+ has no measurable affinity for the phosphate-free, reconstituted mineral core.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Redox reactions of apo mammalian ferritin.

Apo horse spleen ferritin undergoes a 6.3 +/- 0.5 electron redox reaction at -310 mV at pH 6.0-8.5 and 25 degrees C to form reduced apoferritin (apoMFred). Reconstituted ferritin containing up to 50 ferric ions undergoes reduction at the same potential, taking up one electron per ferric ion and six additional electrons by the protein. We propose that apo mammalian ferritin (apoMF) contains six redox centers that can be fully oxidized forming oxidized apoferritin (apoMFox) or fully reduced forming apoMFred. ApoMFred can be prepared conveniently by dithionite or methyl viologen reduction. ApoMFred is slowly oxidized by molecular oxygen but more rapidly by Fe(CN)6(3-) to apoMFox. Fe(III)-cytochrome c readily oxidizes apoMFred to apoMFox with a stoichiometry of 6 Fe(III)-cytochrome c per apoMFred, demonstrating a rapid interprotein electron-transfer reaction. Both redox states of apoMF react with added Fe3+ and Fe2+. Addition of eight Fe2+ to apoMFox under anaerobic conditions produced apoMFred and Fe3+, as evidenced by the presence of a strong g = 4.3 EPR signal. Subsequent addition of bipyridyl produced at least six Fe(bipyd)3(2+) per MF, establishing the reversibility of this internal electron-transfer process between the redox centers of apoMF and bound iron. Incubation of apoMFred with the Fe(3+)-ATP complex under anaerobic conditions resulted in the formation and binding of two Fe2+ and four Fe3+ by the protein. The various redox states formed by the binding of Fe2+ and Fe3+ to apoMFox and apoMFred are proposed and discussed. The yellow color of apoMF appears to be an integral characteristic of the apoMF and is possibly associated with its redox activity.

2,2'-Dipyridyl↗
PubMed1992

Function and CO binding properties of the NiFe complex in carbon monoxide dehydrogenase from Clostridium thermoaceticum.

Adding 1,10-phenanthroline to carbon monoxide dehydrogenase from Clostridium thermoaceticum results in the complete loss of the NiFeC EPR signal and the CO/acetyl-CoA exchange activity. Other EPR signals characteristic of the enzyme (the gav = 1.94 and gav = 1.86 signals) and the CO oxidation activity are completely unaffected by the 1,10-phenanthroline treatment. This indicates that there are two catalytic sites on the enzyme; the NiFe complex is required for catalyzing the exchange and acetyl-CoA synthase reactions, while some other site is responsible for CO oxidation. The strength of CO binding to the NiFe complex was examined by titrating dithionite-reduced enzyme with CO. During the titration, the NiFeC EPR signal developed to a final spin intensity of 0.23 spin/alpha beta. The resulting CO titration curve (NiFeC spins/alpha beta vs CO pha beta) was fitted using two reactions: binding of CO to the oxidized NiFe complex, and reduction of the CO-bound species to a form that exhibits the NiFeC signal. Best fits yielded apparent binding constants between 6000 and 14,000 M-1 (Kd = 70-165 microM). This sizable range is due to uncertainty whether CO binds to all or only a small fraction (approximately 23%) of the NiFe complexes. Reduction of the CO-bound NiFe complex is apparently required to activate it for catalysis. The electron used for this reduction originates from the CO oxidation site, suggesting that delivery of a low-potential electron to the CO-bound NiFe complex is the physiological function of the CO oxidation reaction catalyzed by this enzyme.

Aldehyde Oxidoreductases↗
PubMed1994

Human ferrochelatase is an iron-sulfur protein.

Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed approximately 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40,000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Delayed fluorescence from Fe-S type photosynthetic reaction centers at low redox potential.

Fluorescence kinetics were measured in membranes of a photosystem II-deletion mutant of the cyanobacterium Synechocystis sp. PCC 6803 containing photosystem I as the only reaction center and of the anoxygenic photosynthetic bacterium Heliobacillus mobilis. The measurements were performed under conditions where forward electron transfer to secondary acceptors was inhibited by the strong reductant sodium dithionite. Delayed fluorescence due to recombination of the primary radical pair P+A0- in both species was found to consist of several kinetic components. The longest-lived component had a lifetime of 35 ns in photosystem I and 18 ns in H. mobilis, respectively, which corresponds with the lifetime of the primary radical pair. Delayed fluorescence components with lifetimes of about 4 ns, 1 ns, and 200 ps were also observed in both species and were attributed to relaxations within the radical pair. A standard free energy difference of 0.18 eV was calculated for both species between the relaxed primary radical pair and the excited antenna at room temperature. A value of 0.25 eV was estimated for the free energy difference between the relaxed primary radical pair and the excited primary donor. The temperature dependence of the delayed fluorescence between 25 and 2 degrees C indicated that more than half of the free energy difference is due to enthalpy. Our measurements indicate an overall similarity between the primary electron transfer process in the Fe-S type (or low potential) reaction centers and the (bacterio)pheophytin-quinone type (or high potential) reaction centers found in purple photosynthetic bacteria and photosystem II.

Bacteria↗
PubMed2000

Direct FeS cluster involvement in generation of a radical in lysine 2,3-aminomutase.

Lysine 2,3-aminomutase (KAM) belongs to a class of enzymes that use FeS clusters and S-adenosyl-L-methionine to initiate radical-dependent chemistry. Selenium K-edge X-ray absorption spectroscopic analysis of KAM poised at various stages of catalysis, in the presence of selenomethionine or Se-adenosyl-L-selenomethionine, reveals that the cofactor is cleaved only in the presence of dithionite and the substrate analogue trans-4,5-dehydrolysine. A new Fourier transform peak at 2.7 A, assigned as a Se-Fe interaction, appears concomitant with this cleavage. This is the first demonstration of a direct interaction of S-adenosyl-L-methionine, or its cleavage products, with the FeS cluster in this class of enzymes.

Deoxyadenosines↗
PubMed1982

Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum.

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.

Clostridium↗
PubMed1983

Acyl-CoA oxidase from Candida tropicalis.

Acyl coenzyme A oxidase (acyl-CoA oxidase) has been isolated in good yield from Candida tropicalis pK 233 grown on n-alkanes. Gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and measurement of flavin content suggest that the oxidase is an octamer of Mr 75 000 subunits each containing one flavin. The oxidase yields the red semiquinone form on dithionite or photochemical reduction, slowly forms an N-5 adduct with 0.16 M sulfite at pH 7.4, and is rapidly reduced by borohydride, forming the 3,4-dihydroflavin isomer. The red flavosemiquinone is only kinetically stabilized with respect to disproportionation in the free enzyme but is thermodynamically stabilized on binding enoyl-CoA derivatives. The enzyme is reduced by butyryl-, octanoyl-, and palmitoyl-CoA without formation of prominent long-wavelength bands. Acyl-CoA oxidase and the acyl-CoA dehydrogenases share many similarities in their interaction with CoA derivatives. For example, both enzymes stabilize the anionic radical on binding enoyl-CoA derivatives, both dehydrogenate 2-oxoheptadecyldethio-CoA but cannot utilize S-heptadecyl-CoA, both form long-wavelength bands with CoA persulfide species, and both enzymes are attacked by the suicide substrates 3,4-pentadienoyl-CoA and (methylene-cyclopropyl)acetyl-CoA at the flavin prosthetic group.

Acyl-CoA Oxidase↗
PubMed1984

Identification of a neutral flavin radical and characterization of a second chromophore in Escherichia coli DNA photolyase.

DNA photolyase from Escherichia coli is a blue protein exhibiting absorption maxima at 580, 475, and 384 nm. One of the two chromophores present in this enzyme has been identified as the blue neutral flavin adenine dinucleotide (FAD) radical on the basis, in part, of visible absorption and electron spin resonance (ESR) data. The enzyme-bound radical (epsilon 580 = 3.6 X 10(3) M-1 cm-1) is stable toward O2 or K3Fe(CN)6, is reversibly reduced by dithionite, and is converted to oxidized FAD upon aerobic denaturation. Disproportionation of the radical is observed upon anaerobic denaturation, consistent with an N-5 unsubstituted radical. The absorbance of the enzyme at lambda greater than 500 nm is due only to the FAD radical whereas the band at 384 nm reflects contributions from both the radical and a second chromophore. The latter is labile when protein free a neutral pH (lambda max = 360 nm, k = 5.5 X 10(-2) min -1 +/- O2), a reaction that is readily monitored by the loss of an intense absorption band at 360 nm following enzyme denaturation under conditions where radical oxidation is immediate. This decomposition is pH dependent and the chromophore is stable at acid pH. Native photolyase is fluorescent (emission lambda max = 470 nm, excitation lambda max = 398 nm). An unlikely fluorescent flavin radical can be excluded by the position of the emission maximum. The enzyme fluorescence is attributed to the second chromophore.

Aerobiosis↗
PubMed1985

Kinetics of hemoprotein reduction and interprotein heme transfer.

The transfer of hemin from one protein to another is an event biologically important for the conservation of heme iron. Hemin entering the circulation (or added to serum) is mainly bound by albumin and then transferred to hemopexin [Morgan, W.T., Liem, H.H., Sutor, R.P., & Muller-Eberhard, U. (1976) Biochim. Biophys. Acta 444, 435-445], and we are now investigating which mechanisms may be operative in enhancing this process. The presence of imidazole has been demonstrated to accelerate hemin transfer from albumin to hemopexin [Pasternack, R.F., Gibbs, E.J., Hoeflin, E., Kosar, W.P., Kubera, G., Skowronek, C. A., Wong, N.M., & Muller-Eberhard, U. (1983) Biochemistry 22, 1753-1758]. The present work is an examination of the effect of the reduction of albumin-bound hemin on the rate of its transfer to hemopexin. Hemin (HmIII., ferriprotoporphyrin IX) was reduced to HmII (ferroprotoporphyrin IX) by the addition of sodium dithionite under argon. The reduction kinetics of HmIII to HmII were studied separately in the two complexes: with human serum albumin (HSA), which binds up to 20 mol of heme/mol (the first mole with K congruent to 10(8)), and with hemopexin (HHx), which binds heme equimolarly (K congruent to 10(13)). The rate of reduction of HmIII to HmII on HSA was first order over several half-lives and linearly dependent on [S2O4(2-)]1/2. At [HSA]0/[HmIII] = 3, the kobsd was (5 X 10(-3) + 0.75[S2O4(2-)]1/2, and with [HSA]/[HmIII] approximately 25, the kobsd was (2 X 10(-3)) + 0.25[S2O4(2-)]1/2. The reduction of HmIII to HmII on human hemopexin (HHx) is much more rapid with kobsd = (2.5 X 10(3))[S2O4(2-)]1/2.(ABSTRACT TRUNCATED AT 250 WORDS)

Dithionite↗
PubMed1986

Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases.

Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1986

Oxidation-reduction potentials of butyryl-CoA dehydrogenase.

In order to obtain butyryl-CoA dehydrogenase from Megasphaera elsdenii in pure enough form to perform redox studies, the existing purification procedures first had to be modified and clarified [Engel, P. (1981) Methods Enzymol. 71, 359-366]. These modifications are described, and the previously unpublished spectral properties of the electrophoretically pure CoA-free butyryl-CoA dehydrogenase are presented. In our spectral reductive titration of pure enzyme, we show that although blue neutral flavin radical is stabilized in nonquantitative amounts in dithionite titrations (19%) or in electrochemical reductions mediated by methylviologen (5%), it is not thermodynamically stabilized; therefore, only a midpoint potential for butyryl-CoA dehydrogenase is obtained. The electron-transfer behavior from pH 5.5 to pH 7.0 indicates reversible two-electron transfer accompanied by one proton: EFlox + 2e- + H+ = EFlredH- Em7 = -0.079 V vs. SHE where EFlox is oxidized butyryl-CoA dehydrogenase, EFlredH- is two electron reduced enzyme, and Em7 is the midpoint potential at pH 7.0 at 25 degrees C. Redox data and activity data both indicate that the enzyme loses activity rapidly at pH values above 7.0. The Em7 of the butyryl-CoA dehydrogenase is 40 mV positive of the Em7 of the butyryl-CoA/crotonyl-CoA couple [Gustafson, W. G., Feinberg, B. A., & McFarland, J. T. (1986) J. Biol. Chem. 261, 7733-7741]. Binding of substrate analogue acetoacetyl-CoA caused the potential of butyryl-CoA dehydrogenase to shift 100 mV negative of the free enzyme. The negative shift in potential makes electron transfer from enzyme to substrate more probable, which is consistent with the direction of electron transfer in the bacterial system.(ABSTRACT TRUNCATED AT 250 WORDS)

Butyryl-CoA Dehydrogenase↗
PubMed1987

Redox properties of the quinoprotein methylamine dehydrogenase from paracoccus denitrificans.

Paracoccus denitrificans synthesizes a methylamine dehydrogenase that contains a covalently bound form of pyrroloquinoline quinone as a prosthetic group [Husain, M., & Davison, V.L. (1987) J. Bacteriol. 169, 1712-1717]. Anaerobic reductive titration of this enzyme with dithionite proceeded through a semiquinone intermediate with spectral properties quite distinct from those of the oxidized and reduced species. From these data the molar extinction coefficients were calculated at various wavelengths for the three redox states of this enzyme. The semiquinone was slowly reoxidized under aerobic conditions. The fully reduced enzyme was stable in the presence of oxygen and slowly reoxidized by ferricyanide. Reductive titration of methylamine dehydrogenase with methylamine proceeded directly to the fully reduced form of the enzyme without detectable formation of the semiquinone. Electrochemical titrations of the enzyme yielded an overall midpoint potential value for the two-electron couple (fully oxidized/fully reduced) of 100 +/- 4 mV and an n value of 2.15 +/- 0.15.

Bacterial Proteins↗
PubMed1988

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding.

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzoates↗
PubMed1989

Evidence for the participation of Cys558 and Cys559 at the active site of mercuric reductase.

Mercuric reductase, with FAD and a reducible disulfide at the active site, catalyzes the two-electron reduction of Hg(II) by NADPH. Addition of reducing equivalents rapidly produces a spectrally distinct EH2 form of the enzyme containing oxidized FAD and reduced active site thiols. Formation of EH2 has previously been reported to require only 2 electrons for reduction of the active site disulfide. We present results of anaerobic titrations of mercuric reductase with NADPH and dithionite showing that the equilibrium conversion of oxidized enzyme to EH2 actually requires 2 equiv of reducing agent or 4 electrons. Kinetic studies conducted both at 4 degrees C and at 25 degrees C indicate that reduction of the active site occurs rapidly, as previously reported [Sahlman, L., & Lindskog, S. (1983) Biochem. Biophys. Res. Commun. 117, 231-237]; this is followed by a slower reduction of another redox group via reaction with the active site. Thiol titrations of denatured Eox and EH2 enzyme forms show that an additional disulfide is the group in communication with the active site. [14C]Iodoacetamide labeling experiments demonstrate that the C-terminal residues, Cys558 and Cys559, are involved in this disulfide. The fluorescence, but not the absorbance, of the enzyme-bound FAD was found to be highly dependent on the redox state of the C-terminal thiols. Thus, Eox with Cys558 and Cys559 as thiols exhibits less than 50% of the fluorescence of Eox where these residues are present as a disulfide, indicating that the thiols remain intimately associated with the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1981

Analysis of the iron-sulfur cluster of aconitase by natural and magnetic circular dichroism.

We have examined the iron-sulfur cluster of aconitase, a high-potential iron-sulfur protein, by absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of unactivated aconitase, which is presumably oxidized, is similar to those of reduced two iron-two sulfide ferredoxins but distinct from the MCD of known four iron-four sulfide proteins. The magnitude of the natural CD of unactivated aconitase also suggests the absence of four iron-four sulfur clusters. Reduction of the enzyme with dithionite and activation with the cysteine-ascorbate-ferrous ion activation mixture generate spectra which are significantly different from those of any iron-sulfur protein seen to date. We interpret these results as indicating that aconitase does not contain a four iron-four sulfur cluster generally thought to be characteristic of high-potential iron-sulfur proteins. It could contain a two iron-two sulfur center or some other center such as a cyclic three iron-three sulfur center.

Aconitate Hydratase↗