A mechanistic and electrochemical study of the interaction between dimethyl sulfide dehydrogenase and its electron transfer partner cytochrome c 2

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron–sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c ₂ mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c ₂ accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c ₂), Michaelis constants, K _M,DMS and K _M,cyt of 53 and 21 μM, respectively, were determined at pH 8. Further kinetic analysis revealed a “ping-pong” enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c ₂ immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible Fe^III/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The Fe^III/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c ₂ is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c ₂ effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant of 13(±1) μM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c ₂ as the electron acceptor was found to be approximately 8.3.     

Sulfide-quinone reductase activity in membranes of the chemotrophic bacterium Paracoccus denitrificans GB17

Reduction of exogenous ubiquinone and of cytochromes by sulfide in membranes of the chemotrophic bacterium Paracoccus denitrificans GB17 was studied. For sulfide-ubiquinone reductase activity, K _m values of 26 ± 4 and 3.1 ± 0.6 μM were determined from titrations with sulfide and decyl-ubiquinone, respectively. A maximal rate of up to 0.3 μmol decyl-ubiquinone reduced (mg protein)^–1 min^–1 was estimated. The reaction was sensitive to quinone-analogous inhibitors, but insensitive to cyanide. Reduction of cytochromes by sulfide was monitored with an LED-array spectrophotometer. Under oxic conditions, reduction rates and extents of reduction were lower than those under anoxic conditions. Reoxidation of cytochromes was oxygen-dependent and cyanide-sensitive. The multiphasic behavior of transient reduction of cytochrome b with limiting amounts of sulfide reflects that sulfide, in addition to acting as an electron donor, is a slowly binding inhibitor of cytochrome c oxidase. The initial peak of cytochrome b reduction is dependent on electron flow to an oxidant, either oxygen or ferricyanide, and is stimulated by antimycin A. This oxidant-induced reduction of cytochrome b suggests that electron transport from sulfide in P. denitrificans GB17 employs the cytochrome bc ₁ complex via the quinone pool. Received: 8 April 1998 / Accepted: 29 July 1998     

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180^T) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum. Received: 5 November 1997 / Accepted: 30 March 1998     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (E _m7 = –254 mV) and one low-spin, high-potential (E _m7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c ₅₅₅ as the electron donor, the K _M and V _max for peroxide reduction were 510 ± 100 nM and 425 ± 22 mol ferrocytochrome c ₅₅₅ oxidized min^–1 (mole cytochrome c peroxidase)^–1, respectively. Received: 6 January 1997 / Accepted: 27 May 1997     

Analysis of the activation mechanism of <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase through an electron transfer chain

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an \textE_\textr \textC_\texti^￠ {\text{E}}_{\text{r}} {\text{C}}_{\text{i}}^{\prime } catalytic mechanism. An intermolecular electron transfer rate constant of (4 ± 1) × 10⁵ M⁻¹ s⁻¹ was estimated for both forms of the enzyme, as well as a similar Michaelis–Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.     

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3–0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with E _m,7 of +171 ± 10, +62 ± 10 and –45 ± 13 mV (561–575 nm), and +268 ± 6, +137 ± 10 and –43 ± 12 mV (551–540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (E _m,7≥ +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c ₂ class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60–70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 μM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 μM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c. Received: 8 June 1998 / Accepted: 25 August 1998     

Oxygen consumption and sulfide detoxification in the lugworm Arenicola marina (L.) at different ambient oxygen partial pressures and sulfide concentrations

The lugworm Arenicola marina is a typical inhabitant of intertidal flats. In its L-shaped burrow the animal is exposed to varying concentrations of O₂ and toxic sulfide depending on the tides. The lugworm is able to detoxify sulfide through its oxidation to thiosulfate. When exposed to declining O₂ tensions Arenicola marina reacted as an oxyconformer. In the presence of 25 μmol · l⁻¹ sulfide the respiration was not affected. In contrast, the lugworm consumed significantly less O₂ at any Po₂ in the presence of 200 μmol · l⁻¹ sulfide. Without sulfide anaerobic metabolism started at a Po₂ of approximatedly 10 kPa. Even at high O₂ tensions animals exposed to sulfide produced significantly more anaerobic metabolites compared with the controls. Accordingly the critical value Pc_M, the ambient Po₂ below which anaerobic metabolism starts, was shifted towards normoxia. Since O₂ supply was sufficient for aerobic metabolism, anaerobiosis was induced by sulfide. An influx of sulfide was observed at 25 as well as at 200 μmol · l⁻¹ sulfide. The main product of sulfide detoxification in the lugworm was thiosulfate. Its synthesis increased with ambient Po₂ and depended on the sulfide concentration. Sulfide and thiosulfate were detected in the coelomic fluid, the blood, and the body wall of Arenicola marina. Only about 2% of the ambient O₂ was used for sulfide detoxification at 25 μmol · l⁻¹ sulfide and about 50% at 200 μmol · l⁻¹ sulfide, respectively. Even at the low sulfide concentration Arenicola marina's capacity to detoxify sulfide was too low to maintain a complete aerobic metabolism. Accepted: 19 February 1997     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated

 The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H₂O₂ to H₂O with oxidation of cytochrome c ₅₅₀ and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4′-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca²⁺ is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem. Received: 29 April 1998 / Accepted: 3 September 1998     

Transformation of iron sulfide to greigite by nitrite produced by oil field bacteria

Nitrate, injected into oil fields, can oxidize sulfide formed by sulfate-reducing bacteria (SRB) through the action of nitrate-reducing sulfide-oxidizing bacteria (NR-SOB). When reservoir rock contains siderite (FeCO₃), the sulfide formed is immobilized as iron sulfide minerals, e.g. mackinawite (FeS). The aim of our study was to determine the extent to which oil field NR-SOB can oxidize or transform FeS. Because no NR-SOB capable of growth with FeS were isolated, the well-characterized oil field isolate Sulfurimonas sp. strain CVO was used. When strain CVO was presented with a mixture of chemically formed FeS and dissolved sulfide (HS⁻), it only oxidized the HS⁻. The FeS remained acid soluble and non-magnetic indicating that it was not transformed. In contrast, when the FeS was formed by adding FeCl₂ to a culture of SRB which gradually produced sulfide, precipitating FeS, and to which strain CVO and nitrate were subsequently added, transformation of the FeS to a magnetic, less acid-soluble form was observed. X-ray diffraction and energy-dispersive spectrometry indicated the transformed mineral to be greigite (Fe₃S₄). Addition of nitrite to cultures of SRB, containing microbially formed FeS, was similarly effective. Nitrite reacts chemically with HS⁻ to form polysulfide and sulfur (S⁰), which then transforms SRB-formed FeS to greigite, possibly via a sulfur addition pathway (3FeS + S⁰ → Fe₃S₄). Further chemical transformation to pyrite (FeS₂) is expected at higher temperatures (>60°C). Hence, nitrate injection into oil fields may lead to NR-SOB-mediated and chemical mineral transformations, increasing the sulfide-binding capacity of reservoir rock. Because of mineral volume decreases, these transformations may also increase reservoir injectivity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

Purification and characterization of eugenol dehydrogenase from Pseudomonas fluorescens E118

Pseudomonas fluorescens E118 was isolated from soil as an effective eugenol-degrading organism by a screening using eugenol as enrichment substrate. The first enzyme involved in the degradation of eugenol in this organism, eugenol dehydrogenase, was purified after induction by eugenol, and the purity of the enzyme was shown by SDS-PAGE and gel-permeation HLPC. The enzyme is a heterodimer that consists of a 10-kDa cytochrome c and a 58-kDa subunit. The larger subunit presumably contains flavin, suggesting a flavocytochrome c structure and an electron transfer via flavin and cytochrome c during dehydrogenation. The activity of the purified enzyme depended on the addition of a final electron acceptor such as phenazine methosulfate, 2,6-dichlorophenol-indophenol, cytochrome c, or potassium ferricyanide. The enzyme catalyzed the dehydrogenation of three different 4-hydroxybenzylic structures including the conversion of eugenol to coniferyl alcohol, 4-alkylphenols to 1-(4-hydroxyphenyl)alcohols, and 4-hydroxybenzylalcohols to the corresponding aldehydes. The catalytic and structural similarity between this enzyme and a Penicillium vanillyl-alcohol oxidase and 4-alkylphenol methylhydroxylases from several Pseudomonas species is discussed. Received: 17 June 1998 / Accepted: 12 October 1998     

Chromate reduction by Rhodobacter sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 μM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of R. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30°C. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K _m of 15±1.3 μM CrO₄ ²⁻ and a V _max of 420±50 μmol min⁻¹ mg protein⁻¹ was determined for the enzyme isolated from anaerobically grown cells and 29±6.4 μM CrO₄ ²⁻ and 100±9.6 μmol CrO₄ ²⁻ min⁻¹ mg protein⁻¹ for the one from aerobically grown ones. Journal of Industrial Microbiology & Biotechnology (2000) 25, 198–203. Received 05 January 2000/ Accepted in revised form 27 May 2000     

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A ₂₈₀/A _{417 red}=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: =553,5 nm; =523 nm; =417 nm, while the oxidized form shows a -band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A ₂₈₀/A _{412 ox}=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (=554,5 nm) can be detected spectrophotometrically.Non-common abbreviations HIPIP high-potential iron sulfur protein - APS adenylylsulfate     

Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport

Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H₂ oxidized min^–1 (mg protein)^–1 with benzyl viologen as electron acceptor and an apparent K _m value for H₂ of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 ± 1.7 mol Fe, 11.4 ± 1.4 mol S^2–, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)⁺. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H₂-dependent cytochrome b reduction at a rate of 0.18 nmol min^–1 (mg protein)^–1. Received: 7 September 1995 / Accepted: 4 December 1995     

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c 3

 Reduction of the haems in tetrahaem cytochromes c ₃ is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c ₃ form a structural motif that is present in all cytochromes c ₃ and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c ₃. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c ₃. NMR studies of F20I cytochrome c ₃ demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c ₃ backbone, which is also very close to haem I, had no effect on the network of cooperativities. Received: 25 June 1996 / Accepted: 26 August 1996     

Artefacts induced on <Emphasis Type="Italic">c</Emphasis>-type haem proteins by electrode surfaces

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met–His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c ₃ from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.