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PubMed1994

Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.

The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes.

Ascorbic Acid↗
PubMed1983

Light-dependent nitration of bacteriorhodopsin.

Purple membranes were treated with tetranitromethane to modify tyrosine residues of bacteriorhodopsin. At pH 8.0, nitration is shown to be affected by illumination during the modification. Amino acid analysis revealed about 0.7 residues nitrated if reaction was in the dark while about 2.0 tyrosines were modified if illumination greater than 540 nm was provided. Tryptophan was unaffected under both conditions. Light-dependent nitration caused a blue shift of the absorbance maximum of bacteriorhodopsin from 568 to 530 nm while no chromophore shift was observed for the dark-modified preparation. Both preparations show an absorption band at 360 nm indicative of the presence of nitrotyrosines. Reduction by dithionite eliminated the pH-dependent changes associated with the 360-nm nitrotyrosine band. Circular dichroism spectra indicate that interactions between neighboring chromophores are altered concomitant with the blue shift of the absorbance maximum by nitration. These studies show that light is required for the nitration of the tyrosine residue, and that Tyr 26 (H. D. Lemke and D. Oesterhelt (1981) Eur. J. Biochem. 115, 595-604) is probably responsible for the blue shift of the absorbance maximum. The intrinsic fluorescence and photocycle kinetics of the tyrosine-modified preparation and reduction of nitrotyrosine by dithionite were studied. In dark modification, only pH-dependent dithionite-reducible nitrotyrosines were produced. It is concluded that surface tyrosines probably do not directly participate in the proton-translocation events coupled to the photocycle of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1975

Characterization of the respiratory nitrate reductase of Klebsiella aerogenes as a molybdenum-containing iron-sulfur enzyme.

1. In respiratory nitrate reductase I of Klebsiella aerogenes, 0.24 atom of molybdenum, eight iron-sulfur groups and four tightly bound, non-heme iron atoms per molecule of enzyme (Mr 260 000) are found. 2. EPR spectra at 83 degrees K of oxidized and reduced nitrate reductase I show complex lines at g = 2.02 and g = 1.98, which are more intense in the reduced than in the oxidized enzyme. The resonances, the shape and intensity of which are rather temperature insensitive, are attributed to two species of paramagnetic molybdenum. In dithionite-reduced enzyme all these lines are saturated at the same microwave power of 15 mW. This is not the case in oxidized enzyme, where the resonance at g = 2.02 is hard to saturate. Addition of nitrate to dithionite-reduced reductase I decreases the intensity of the EPR lines to about that of oxidized enzyme. The participation of molybdenum in the electron transfer process has been discussed. 3. At 18 degrees K the oxidized enzyme exhibits an axial-symmetrical signal with g parallel = 2.10 and g = 2.03, and a signal with unknown symmetry at g = 2.015. Upon reduction by dithionite, a ferredoxin type of signal is observed with g values at 2.05, 1.95 and 1.88, while the g = 2.015 signal disappears. Reoxidation by nitrate causes a concomitant disappearance of the ferredoxin type of signal and reappearance of the g = 2.015 signal; hence iron-sulfur centres participate in the transfer of electrons to nitrate. 4. Nitrate reductase II, containing only two (Mr 117 000 and 57 000) of the three subunits found in nitrate reductase I and lacking the tightly bound iron, does not exhibit the axial-symmetrical signal (g = 2.10 and 2.03). Thus, it suggested that this signal in nitrate reductase I stems from an iron centre in the low-molecular weight subunit (Mr 52 000). 5. Inhibition studies confirm the participation of metals in the transfer of electrons from reduced benzylviologen to nitrate and show that the binding sites for these substrates are different.

Binding Sites↗
PubMed1990

Spectroscopic studies of partially reduced forms of Wolinella succinogenes nitrite reductase.

Reductive titrations of the dissimilatory hexa-haem nitrite reductase, Wolinella succinogenes, with methyl viologen semiquinone (MV) and sodium dithionite, have been followed at room temperature by absorption, natural (CD) and magnetic circular dichroism (MCD) spectroscopies and at liquid helium temperature by electron paramagnetic resonance (EPR) and MCD spectroscopies. The nature of the reduced enzyme depends on the reductant employed. At room temperature a single high-spin ferrous haem, observed by MCD after reduction with MV, is absent from dithionite reduced samples. It is suggested that a product of dithionite oxidation becomes bound with high affinity to the reduced state of the enzyme causing the ferrous haem to become low-spin. The site occupied is likely to be the substrate binding haem. The course of the titration with MV at room temperature shows the reduction of high-spin ferric to high-spin ferrous haem. Since the EPR spectrum reveals the presence of an unusual high-low spin ferric haem pair in the oxidised state we propose that the active site of the enzyme is a novel haem pair consisting of one high (5-coordinate) and one low-spin (6 coordinate) haem, magnetically coupled and possibly bridged by a histidinate ligand.

Bacteroidaceae↗
PubMed1987

A study of the catalase monomer produced by lyophilization.

Lyophilization of Dounce and Mourtzikos beef liver catalase (Prep. Biochem. 11 (1981) 501-523) under specified conditions produced conformationally altered but not completely denatured catalase monomer which retained both significant catalatic activity and peroxidatic activity towards ethanol. The same lyophilization procedure used with Sigma Co. catalase produced a mixture of conformationally altered catalase monomer and conformationally altered tetramer which showed still higher catalatic and peroxidatic activities; this was attributed to the presence of the altered tetramer. The catalase monomer obtained by the use of Dounce and Mourtzikos catalase is completely reducible by dithionite, as shown by the two-banded spectrum of the reduced material, but apparently retains enough of its native conformation to show some enzymatic activity, since the fully denatured monomer shows no catalatic or peroxidatic activity towards ethanol. The conformationally altered catalase tetramer, which shows more enzymatic activity than the monomer, evidently retains a higher proportion of its native conformation than the monomer, but still appears to be fully reducible with dithionite. Horseradish peroxidase after reduction with dithionite shows spectral bands at positions close to those of reduced lyophilized catalase, but the relative band heights and contours are different. A possible explanation for the observed differences in lyophilization products depending on the starting material (Sigma Co. catalase versus catalase of Dounce and Mourtzikos) is presented.

Animals↗
PubMed2001

Dynamics of membrane penetration of the fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group attached to an acyl chain of phosphatidylcholine.

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Regeneration of surfactant-modified zeolite after saturation with chromate and perchloroethylene.

Surfactant-modified zeolites (SMZ) have drawn recent attention as sorbents due to their removal of multiple types of contaminants and low material cost. However, like most sorbents, SMZ has a finite sorption capacity for different contaminants. The potential applications, economics, and efficiency of SMZ as a sorbent are related to the ability to regenerate the material. This paper reports several methods to regenerate chromate- and perchloroethylene-saturated SMZ. Regeneration of chromate-saturated SMZ was achieved by flushing with a sodium carbonate/sodium hydroxide solution. However, this high-pH solution increased the counterion competition for chromate sorption sites and decreased the chromate sorption capacity of the regenerated SMZ. As an alternative regeneration method, chromate sorbed to SMZ was reduced to Cr(III) in situ using sodium dithionite solution. Although reduction with dithionite restored the chromate sorption maximum, the chromate sorption intensity was lowered, possibly due to the competition by sulfate (generated from oxidation of dithionite) for chromate sorption sites. Carbonate-regenerated SMZ showed no loss of sorption affinity for perchloroethylene (PCE) compared to virgin SMZ. Air sparging of PCE-saturated SMZ fully regenerated the SMZ. The results show that it is possible to regenerate and re-use SMZ following saturation with anionic or nonpolar organic contaminants.

Chromates↗
PubMed1995

Intermolecular electron transfer from substrate-reduced methylamine dehydrogenase to amicyanin is linked to proton transfer.

Within the methylamine dehydrogenase-amicyanin complex, intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) and copper. The ET reactions from two chemically distinct reduced forms of TTQ were studied. The quinol form of TTQ was generated by reduction by dithionite. An aminoquinol form of TTQ, in which an amino group displaces the carbonyl oxygen, was generated by reduction by the substrate methylamine. Thermodynamic analysis of the ET reactions suggested that the ET event was rate-limiting for the redox reaction between quinol TTQ and copper, but not for the ET reaction from aminoquinol TTQ to copper. Solvent kinetic isotope effect studies indicated that proton transfer was involved in the rate-limiting reaction step for the ET from the substrate-reduced enzyme, but not the dithionite-reduced enzyme. Solvent deuterium kinetic isotope effects of 1.5 and 12.2 were obtained, respectively, for the ET reactions from dithionite-reduced and substrate-reduced methylamine dehydrogenase. These results demonstrate that application of ET theory to the analysis of thermodynamic data for the intermolecular protein ET reactions can potentially be used to distinguish between true ET reactions and those which are gated or attenuated by adiabatic events. Kinetic models are presented to explain how the incorporation of the substrate-derived amino group into TTQ may alter the rate-limiting step for ET.

Bacterial Proteins↗
PubMed1991

Formation of prostaglandin synthase-iron-nitrosoalkane inhibitory complexes upon in situ oxidation of N-substituted hydroxylamines.

Various N-alkylhydroxylamines such as N-hydroxyamphetamine react with prostaglandin synthase (PGHS) from sheep seminal vesicles, with the formation of new complexes characterized by a Soret peak around 421 nm. These complexes are very stable toward O2 or dithionite but are destroyed upon oxidation by Fe(CN)6K3 with regeneration of starting PGHS-FeIII. Their spectral characteristics, chemical properties, and routes of formation (either by direct oxidation of RNHOH or by in situ reduction of RNO2 in the presence of dithionite) are very similar to those previously reported for nitrosoalkane complexes of hemoglobin-, myoglobin-, and cytochrome P-450-FeII. Their FeII-N(O)R structure was completely confirmed in the case of N-hydroxyamphetamine, both by extraction of the heme complex by butanone and by identification to authentic protoporphyrin IX-FeII-N(O)-amphetamine, and by insertion of this authentic complex into apoPGHS. Phenylhydroxylamine also reacts with PGHS-FeIII to give a PGHS-FeII-N(O)Ph complex which is not stable in the presence of dithionite because of its weaker PGHS-FeII-N(O)R bond when compared to PGHS-FeII-nitrosoalkane complexes. The ability of various N-alkylhydroxylamines to form PGHS-FeII-N(O)R complexes greatly depends upon their hydrophobicity. Actually, CH3NHOH and C2H5NHOH are totally inactive whereas about 10 molar excess of N-hydroxyamphetamine and C6H5NHOH already lead to 50% complex formation. This is in favor of an hydrophobic environment of the heme in PGHS. Finally, PGHS engaged in such FeII-nitrosoalkane complexes completely loses its dioxygenase activity, suggesting that N-substituted hydroxylamines or compounds that can be metabolized in vivo to give such hydroxylamines could act as strong PGHS inhibitors.

Animals↗
PubMed1989

DNA cross-linking by intermediates in the mitomycin activation cascade.

We have assayed the cross-linking of oligonucleotides containing repeated mitomycin-reactive CpG sites in order to assess the factors that enhance activation of the carbamoyl function at C10, yielding efficient mitomycin cross-linking. Drugs studied include mitomycin C (MC), N-methylmitomycin A (NMA), and the aziridinomitosene of NMA (MS). Drugs were reduced both by catalytic hydrogenation and by diothionite. We find that cross-linking by fully reduced NMA can be increased severalfold by addition of either excess dithionite reductant or the oxidant FeCl3. Enhancement by FeCl3 is not seen with MC or MS, but excess dithionite increases cross-linking by all three compounds. We explain the action of Fe3+ by postulating production of the semiquinone of the monoadduct of mitomycin reacted at the C1-position; according to this mechanism, departure of the carbamate from C10 is more efficient for the semiquinone than for the hydroquinone. However, our results imply that the hydroquinone can also function as a cross-linking agent. Excess dithionite, beyond that required for stoichiometric reduction, activates the carbamate 2-3-fold for cross-linking. We find that the fully reduced leucoaziridinomitosene is highly unstable in solution, yet it produces efficient cross-liking. Hence, this compound is highly reactive in DNA alkylation and a good candidate for the role of primary alkylating agent.

Base Sequence↗
PubMed2002

Alcaligenes xylosoxidans dissimilatory nitrite reductase: alanine substitution of the surface-exposed histidine 139l ligand of the type 1 copper center prevents electron transfer to the catalytic center.

Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g(1) = 2.347 and A(1) = 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g(1) = 2.29 and A(1) = 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E degree = +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K(2)IrCl(6) (E degree = +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g(1) = 2.20 and A(1) = 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K(2)IrCl(6) by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least approximately 470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO(4) was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g(1) = 2.226 and A(1) = 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.

Alanine↗
PubMed2004

Thermodynamic basis of electron transfer in dihydroorotate dehydrogenase B from Lactococcus lactis: analysis by potentiometry, EPR spectroscopy, and ENDOR spectroscopy.

Dihydroorotate dehydrogenase B (DHODB) is a complex iron-sulfur flavoprotein that catalyzes the conversion of dihydroorotate to orotate and the reduction of NAD(+). The enzyme is a dimer of heterodimers containing an FMN, an FAD, and a 2Fe-2S center. UV-visible, EPR, and ENDOR spectroscopies have been used to determine the reduction potentials of the flavins and the 2Fe-2S center and to characterize radicals and their interactions. Reductive titration using dithionite indicates a five-electron capacity for DHODB. The midpoint reduction potential of the 2Fe-2S center (-212 +/- 3 mV) was determined from analysis of absorption data at 540 nm, where absorption contributions from the two flavins are small. The midpoint reduction potentials of the oxidized/semiquinone (E(1)) and semiquinone/hydroquinone (E(2)) couples for the FMN (E(1) = -301 +/- 6 mV; E(2) = -252 +/- 8 mV) and FAD (E(1) = -312 +/- 6 mV; E(2) = -297 +/- 5 mV) were determined from analysis of spectral changes at 630 nm. Corresponding values for the midpoint reduction potentials for FMN (E(1) = -298 +/- 4 mV; E(2) = -259 +/- 5 mV) in the isolated catalytic subunit (subunit D, which lacks the 2Fe-2S center and FAD) are consistent with the values determined for the FMN couples in DHODB. During reductive titration of DHODB, small amounts of the neutral blue semiquinone are observed at approximately 630 nm, consistent with the measured midpoint reduction potentials of the flavins. An ENDOR spectrum of substrate-reduced DHODB identifies hyperfine couplings to proton nuclei similar to those recorded for the blue semiquinone of free flavins in aqueous solution, thus confirming the presence of this species in DHODB. Spectral features observed during EPR spectroscopy of dithionite-reduced DHODB are consistent with the midpoint reduction potentials determined using UV-visible spectroscopy and further identify an unusual EPR signal with very small rhombic anisotropy and g values of 2.02, 1.99, and 1.96. This unusual signal is assigned to the formation of a spin interacting state between the FMN semiquinone species and the reduced 2Fe-2S center. Reduction of DHODB using an excess of NADH or dihydroorotate produces EPR spectra that are distinct from those produced by dithionite. From potentiometric studies, the reduction of the 2Fe-2S center and the reduction of the FMN occur concomitantly. The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulfur flavoprotein.

Dihydroorotate Dehydrogenase↗
PubMed1997

Effects of mutation of the conserved lysine-362 in cytochrome c oxidase from Rhodobacter sphaeroides.

We describe the effects of a mutation, K362M, of the conserved lysine in cytochrome c oxidase from Rhodobacter sphaeroides, a residue located in a putative proton channel that may convey substrate protons to the binuclear center. Spectra of the "as prepared", ferricyanide-oxidized, and dithionite-reduced forms of the mutant protein confirm that the redox centers remain intact. Ligand binding kinetics of the ferricyanide-oxidized enzyme and of the dithionite-reducible fraction are similar to those of the wild type, indicating that the K channel is not the major route for CO, cyanide, formate, or peroxide entry into the structure. Protonation of the lysine residue is not redox-linked to heme a or CuB as judged from the essentially unaltered midpoint potentials of these centers in the cyanide-ligated enzyme. A difficulty in electron transfer from heme a to the binuclear center is indicated by the slow and only partial reduction of heme a3 by dithionite or ferrocytochrome c and by the presence of some reduced heme a in the as prepared mutant enzyme and under steady-state conditions. Further characterization of the K362M enzyme in the steady state shows that up to one electron, but not two, can enter the binuclear center easily. It is this inability to form the two-electron-reduced, oxygen-reactive R state that prevents activity. A model is proposed where the K channel serves as a dielectric well of high dielectric strength and low proton conductivity, rather than as a pathway for proton entry to the binuclear center. The function of this structure would be to decrease the cost of introducing a transiently uncompensated charge into the binuclear center prior to formation of a stable, charge-compensated R-state.

Amino Acid Sequence↗
PubMed1999

Effects of copper and zinc ions on photosystem II studied by EPR spectroscopy.

The effect of Zn(2+) or Cu(2+) ions on Mn-depleted photosystem II (PS II) has been investigated using EPR spectroscopy. In Zn(2+)-treated and Cu(2+)-treated PS II, chemical reduction with sodium dithionite gives rise to a signal attributed to the plastosemiquinone, Q(A)(*)(-), the usual interaction with the non-heme iron being lost. The signal was identified by Q-band EPR spectroscopy which partially resolves the typical g-anisotropy of the semiquinone anion radical. Illumination at 200 K of the unreduced samples gives rise to a single organic free radical in Cu(2+)-treated PS II, and this is assigned to a monomeric chlorophyll cation radical, Chl a(*)(+), based on its (1)H-ENDOR spectrum. The Zn(2+)-treated PS II under the same conditions gives rise to two radical signals present in equal amounts and attributed to the Chl a(*)(+) and the Q(A)(*)(-) formed by light-induced charge separation. When the Cu(2+)-treated PS II is reduced by sodium ascorbate, at >/=77 K electron donation eliminates the donor-side radical leaving the Q(A)(*)(-) EPR signal. The data are explained as follows: (1) Cu(2+) and Zn(2+) have similar effects on PS II (although higher concentrations of Zn(2+) are required) causing the displacement of the non-heme Fe(2+). (2) In both cases chlorophyll is the electron donor at 200 K. It is proposed that the lack of a light-induced Q(A)(*)(-) signal in the unreduced Cu(2+)-treated sample is due to Cu(2+) acting as an electron acceptor from Q(A)(*)(-) at low temperature, forming the Cu(+) state and leaving the electron donor radical Chl a(*)(+) detectable by EPR. (3) The Cu(2+) in PS II is chemically reducible by ascorbate prior to illumination, and the metal can therefore no longer act as an electron acceptor; thus Q(A)(*)(-) is generated by illumination in such samples. (4) With dithionite, both the Cu(2+) and the quinone are reduced resulting in the presence of Q(A)(*)(-) in the dark. The suggested high redox potential of Cu(2+) when in the Fe(2+) site in PS II is in contrast to the situation in the bacterial reaction center where it has been shown in earlier work that the Cu(2+) is unreduced by dithionite. It cannot be ruled out however that Q(A)-Cu(2+) is formed and a magnetic interaction is responsible for the lack of the Q(A)(-) signal when no exogenous reductant is present. With this alternative possibility, the effects of reductants would be explained as the loss of Cu(2+) (due to formation of Cu(+)) leading to loss of the Cu(2+) from the Fe(2+) site due to the binding equilibrium. The quite different binding and redox behavior of the metal in the iron site in PS II compared to that of the bacterial reaction center is presumably a further reflection of the differences in the coordination of the iron in the two systems.

2,6-Dichloroindophenol↗
PubMed1999

Models for molybdenum coordination during the catalytic cycle of periplasmic nitrate reductase from Paracoccus denitrificans derived from EPR and EXAFS spectroscopy.

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.

Azides↗
PubMed2000

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters.

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.

Catalysis↗
PubMed2004

Carbon monoxide induced decomposition of the active site [Ni-4Fe-5S] cluster of CO dehydrogenase.

During the past two years, crystal structures of Cu- and Mo-containing carbon monoxide dehydrogenases (CODHs) and Ni- and Fe-containing CODHs have been reported. The active site of CODHs from anaerobic bacteria (cluster C) is composed of Ni, Fe, and S for which crystallographic studies of the enzymes from Carboxydothermus hydrogenoformans, Rhodospirillum rubrum, and Moorella thermoaceticarevealed structural similarities in the overall protein fold but showed substantial differences in the essential Ni coordination environment. The [Ni-4Fe-5S] cluster C in the fully catalytically competent dithionite-reduced CODH II from C. hydrogenoformans (CODHII(Ch)) at 1.6 A resolution contains a characteristic mu(2)-sulfido ligand between Ni and Fe1, resulting in a square-planar ligand arrangement with four S-ligands at the Ni ion. In contrast, the [Ni-4Fe-4S] clusters C in CO-treated CODH from R. rubrum resolved at 2.8 A and in CO-treated acetyl-CoA synthase/CODH complex from M. thermoacetica at 2.2 and 1.9 A resolution, respectively, do not contain the mu(2)-sulfido ligand between Ni and Fe1 and display dissimilar geometries at the Ni ion. The [Ni-4Fe-4S] cluster is composed of a cubane [Ni-3Fe-4S] cluster linked to a mononuclear Fe site. The described coordination geometries of the Ni ion in the [Ni-4Fe-4S] cluster of R. rubrum and M. thermoacetica deviate from the square-planar ligand geometry in the [Ni-4Fe-5S] cluster C of CODHII(Ch). In addition, the latter was converted into a [Ni-4Fe-4S] cluster under specific conditions. The objective of this study was to elucidate the relationship between the structure of cluster C in CODHII(Ch) and the functionality of the protein. We have determined the CO oxidation activity of CODHII(Ch) under different conditions of crystallization, prepared crystals of the enzyme in the presence of dithiothreitol or dithionite as reducing agents under an atmosphere of N(2) or CO, and solved the corresponding structures at 1.1 to 1.6 A resolutions. Fully active CODHII(Ch) obtained after incubation of the enzyme with dithionite under N(2) revealed the [Ni-4Fe-5S] cluster. Short treatment of the enzyme with CO in the presence of dithiothreitol resulted in a catalytically competent CODHII(Ch) with a CO-reduced [Ni-4Fe-5S] cluster, but a prolonged treatment with CO caused the loss of CO-oxidizing activity and revealed a [Ni-4Fe-4S] cluster, which did not contain a mu(2)-S. These data suggest that the [Ni-4Fe-4S] cluster of CODHII(Ch) is an inactivated decomposition product originating from the [Ni-4Fe-5S] cluster.

Aldehyde Oxidoreductases↗
PubMed2002

Renewable-surface sol-gel derived carbon ceramic electrode fabricated by [Ru(bpy)(tpy)Cl]PF6 and its application as an amperometric sensor for sulfide and sulfur oxoanions.

A highly sensitive and fast responding sensor for the determination of thiosulfate, sulfite, sulfide and dithionite is described. It consists of a chemically modified carbon ceramic composite electrode (CCE) containing [Ru(bpy)(tpy)Cl]PF6 complex that was constructed by the sol-gel technique. A reversible redox couple of Ru(II)/Ru(III) was observed as a solute in acetonitrile solution and as a component of carbon based conducting composite electrode. Electrochemical behavior and stability of modified CCE were investigated by cyclic voltametry, the apparent electron transfer rate constant (kappa(S)) and transfer coefficient (a) were determined by cyclic voltametry which were about 28 s(-1) and 0.43 respectively. Electrocatalytic oxidation of S(2-), SO3(2-), S2O4(2-) and S2O3(2-) were effective at the modified electrode at significantly reduced overpotentials and in the pH range 1-11. Optimum pH values for amperometric detection of thiosulfate, dithionite, sulfide and sulfite are 7, 9, 2 and 2. Under the optimized conditions the calibration curves are linear in the concentration ranges 1-500, 3-80, 2-90 and 1-100 microM for S2O3(2-), SO3(2-), S2- and S2O4(2-) determination. The detection limit (signal to noise is 3) and sensitivity are 0.5 and 12, 2.8 and 6, 1.6 and 8, and 0.65 microM and 80 nA microM(-1) for thiosulfate, sulfite, sulfide and dithionite detection. The modified carbon ceramic electrode doped with Ru-complex shows good reproducibility, a short response time (t < 2 s), remarkable long term stability (> 6 month) and especially good surface renewability by simple mechanical polishing (RSD for eight successive polishing is 2%). The advantages of this sulfur compound amperometric detector based on ruthenium doped CCE are high sensitivity, inherent stability at a broader pH range, excellent catalytic activity, less expense and simplicity of preparation in comparison with recently published papers. This sensor can be used as a chromatographic detector for analysis of sulfur derivatives.

Journal Article↗