Models for Proton-Coupled Electron Transfer in Photosystem II

The coupling of proton and electron transfers is a key part of the chemistry of photosynthesis. The oxidative side of photosystem II (PS II) in particular seems to involve a number of proton-coupled electron transfer (PCET) steps in the S-state transitions. This mini-review presents an overview of recent studies of PCET model systems in the authors’ laboratory. PCET is defined as a chemical reaction involving concerted transfer of one electron and one proton. These are thus distinguished from stepwise pathways involving initial electron transfer (ET) or initial proton transfer (PT). Hydrogen atom transfer (HAT) reactions are one class of PCET, in which H⁺ and e ⁻ are transferred from one reagent to another: AH+B→A+BH, roughly along the same path. Rate constants for many HAT reactions are found to be well predicted by the thermochemistry of hydrogen transfer and by Marcus Theory. This includes organic HAT reactions and reactions of iron-tris(α-diimine) and manganese-(μ-oxo) complexes. In PS II, HAT has been proposed as the mechanism by which the tyrosine Z radical (Y_Z) oxidizes the manganese cluster (the oxygen evolving complex, OEC). Another class of PCET reactions involves transfer of H⁺ and e ⁻ in different directions, for instance when the proton and electron acceptors are different reagents, as in AH–B+C⁺→A–HB⁺+C. The oxidation of Y_Z by the chlorophyll P680 + has been suggested to occur by this mechanism. Models for this process – the oxidation of phenols with a pendent base – are described. The oxidation of the OEC by Y_Z could also occur by this second class of PCET reactions, involving an Mn–O–H fragment of the OEC. Initial attempts to model such a process using ruthenium-aquo complexes are described. An erratum to this article can be found at     

Effects of charged peptides on electron transfer from [Fe(CN)6]4– to cytochrome c or plastocyanin

 Interactions of charged peptides, such as aspartic acid peptides (Aspptds) and lysine peptides (Lysptds), with cytochrome c (cyt c) or plastocyanin (PC) have been studied by measuring electron transfer between [Fe(CN)₆]^4– and cyt c or PC in the presence of these peptides. Aspptds, up to penta-aspartic acid, served as competitive inhibitors of electron transfer from [Fe(CN)₆]^4– to oxidized cyt c, while Lysptds, up to penta-lysine, promoted electron transfer from [Fe(CN)₆]^4– to oxidized PC. The electron transfer inhibitory effects of Aspptds are explained as competitive inhibition due to neutralization of the positively charged amino acid residues at the surface of cyt c by electrostatic interactions, whereas the electron transfer promoting effects of Lysptds may be due to formation of PC·Lysptd or Lysptd·[Fe(CN)₆]^4– complexes subsequently forming an electron transferring complex, PC·Lysptd·[Fe(CN)₆]^4–, without repulsion of the negative charges. The inhibitory effect of Aspptds and promotional effect of Lysptds became significant as the net charge or concentration of the peptides increased. The promotional effects of Lysptds decreased as the net charge of the PC negative patch was decreased by mutagenesis. Thus, charged peptides may serve as a probe for investigation of the molecular recognition character of proteins. Received: 19 May 1998 / Accepted: 27 July 1998     

Kinetic studies of electron transfer in the cyt b 5 : metHb system

 The kinetics of methemoglobin reduction by cytochrome b ₅ has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k _f = 2.44×10⁴ M^–1 s^–1 and a reverse rate constant k _b = 540 M^–1s^–1 have been observed at 10 mm, pH 6.20, 25  °C. The ratio k _f/k _b = k _eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b ₅ and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b ₅ to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin. Received: 20 February 1996 / Accepted: 4 June 1996     

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.     

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B_A⁻ and β⁻, where β is a bacteriochlorophyll substituting the native bacteriopheophytin H_A) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin H_B in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm⁻¹. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or B_A with accumulation of the P⁺β⁻ and/or P⁺B_A⁻ states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼150 cm⁻¹ and distinct low-frequency oscillations at 20–100 cm⁻¹; also, the amplitude of the oscillations at 150 cm⁻¹ is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm⁻¹ mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P⁺H_B⁻ state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.     

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum (Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k _app values, 176.9±25.0 min⁻¹ in the case of the Tp/Tp electron transfer, 31.8±3.6 min⁻¹ for the Dg/Dg electron transfer, and 6.9±1.3 min⁻¹ for Dv/Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron–sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k _app value of 108.8±12.0 min⁻¹, and was then assigned as the potential physiological electron donor in this organism.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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     

Carbon-nanotube-modified electrodes for the direct bioelectrochemistry of pseudoazurin

The bioelectrochemistry of the blue copper protein, pseudoazurin, at glassy carbon and platinum electrodes that were modified with single-wall carbon nanotubes (SWNTs) was investigated by multiple scan rate cyclic voltammetry. The protein showed reversible electrochemical behavior at both bare glassy carbon electrodes (GCEs) and SWNT-modified GCEs (SWNT|GCEs); however, direct electrochemistry was not observed at any of the platinum electrodes. The effect of the carbon nanotubes at the GCE was to amplify the current response 1000-fold (nA at bare GCE to μA at SWNT|GCE), increase the apparent diffusion coefficient D _app of the solution-borne protein by three orders of magnitude, from 1.35 × 10⁻¹¹ at bare GCE to 7.06 × 10⁻⁸ cm² s-1 at SWNT|GCE, and increase the heterogeneous electron transfer rate constant k _s threefold, from 1.7 × 10⁻² cm s⁻¹ at bare GCE to 5.3 × 10⁻² cm s⁻¹ at SWNT|GCE. Pseudoazurin was also found to spontaneously adsorb onto the nanotube-modified GCE surface. Well-resolved voltammograms indicating quasi-reversible faradaic responses were obtained for the adsorbed protein in phosphate buffer, with I _pc and I _pa values now greater than corresponding values for solution-borne pseudoazurin at SWNT|GCEs and with significantly reduced ΔE _p values. The largest electron transfer rate constant of 1.7 × 10⁻¹ cm s⁻¹ was achieved with adsorbed pseudoazurin at the SWNT|GCE surface in deaerated buffer solution consistent with its presumed role in anaerobic respiration of some bacteria.     

Energization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> membrane vesicles increases catalytic activity of succinate: Menaquinone oxidoreductase

In this work, high ΔμH⁺-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells. Deenergization of the membrane vesicles with ionophores or alamethicin brings about an almost complete inhibition of succinate oxidation. An apparent K _m for succinate during the energy-dependent succinate oxidase activity of the vesicles (2.2 mM) is higher by an order of magnitude than the K _m value measured for the energy-independent reduction of 2,6-dichlorophenol indophenol. The data reveal critical importance of ΔμH⁺ for maintaining active electron transfer by succinate:menaquinone oxidoreductase. The role of ΔμH⁺ might consist in providing energy for thermodynamically unfavorable menaquinone reduction by succinate by virtue of transmembrane electron transport within the enzyme down the electric field; alternatively, ΔμH⁺ could play a regulatory role by maintaining the electroneutrally operating enzyme in a catalytically active conformation.     

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     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c 7 family

Cytochromes c ₇ are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins—phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c ₇ family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of ¹H–¹⁵N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.     

A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM⁻¹ cm⁻¹) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K _M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K _M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k _obs1 = 4.7 s⁻¹, k _obs2 = 1.9 s⁻¹) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k _obs3 = 6.10 × 10⁻² s⁻¹, k _obs4 = 2.18 × 10⁻² s⁻¹). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.     

A charge transfer process in the visual pigments

A physical model that incorporates all the experimental information on the formation of the visual pigment rhodopsin is presented. The visual pigments consist of a chromophore bound to an appropriate protein. Thus rhodopsin (λ_m 505 mμ) is formed by a Schiff’s base linkage C₁₉H₂₇CH=NH⁺-opsin (λ_m 440 mμ) between 11-cis retinal (λ_m 380 mμ) and the protein opsin (λ_m 280 mμ). It is found that there exists a red shift in the spectrum of rhodopsin from the Schiff’s base. The model brings an explanation for this red shift. It is shown that such a shift may be due to a charge transfer process (R. S. Mulliken,J. Am. Chem. Soc.,74, 811–824, 1952) between an electron at the double bond of carbons C₁₁−C₁₂ and an atomic orbital of the sulphur present in cysteine. This provides an explanation of the presence of SH-groups in the protein after the absorption of light. A one-electron approximation is used and the dipole momentμ _NV ; hence, the oscillator strengthf of the transitionNV is estimated and compared with the experimentally determined extinction coefficient ∈_m by mixing 3.5×10⁻³ M of 11-cis retinal with 8.3×10⁻⁵ M of cysteine at pH ranges 6 through 8. Reasonable agreement is found. Solvent, concentration and temperature dependence are shown also.     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular orbital study of the hydroxylation of benzene and monofluorobenzene catalysed by iron-oxo porphyrin π cation radical complexes

 The reaction mechanism for the hydroxylation of benzene and monofluorobenzene, catalysed by a ferryl-oxo porphyrin cation radical complex (compound) is described by electronic structure calculations in local spin density approximation. The active site of the enzyme is modelled as a six-coordinated (Por⁺)Fe(IV)O a_2u complex with imidazole or H₃CS^– as the axial ligand. The substrates under study are benzene and fluorobenzene, with the site of attack in para, meta and ortho position with respect to F. Two reaction pathways are investigated, with direct oxygen attack leading to a tetrahedral intermediate and arene oxide formation as a primary reaction step. The calculations show that the arene oxide pathway is distinctly less probable, that hydroxylation by an H₃CS^––coordinated complex is energetically favoured compared with imidazole, and that the para position with respect to F is the preferred site for hydroxylation. A partial electron transfer from the substrate to the porphyrin during the reaction is obtained in all cases. The resulting charge distribution and spin density of the substrates reveal the transition state as a combination of a cation and a radical σ-adduct intermediate with slightly more radical character in the case of H₃CS^– as axial ligand. A detailed analysis of the orbital interactions along the reaction pathway yields basically different mechanisms for the modes of substrate–porphyrin electron transfer and rupture of the Fe–O bond. In the imidazole-coordinated complex an antibonding π*(Fe–O) orbital is populated, whereas in the H₃CS^––coordinated system a shift of electron density occurs from the Fe–O bond region into the Fe–S bond. Received: 1 July 1995 / Accepted: 18 December 1995     

A novel membrane-bound flavocytochrome c sulfide dehydrogenase from the colourless sulfur bacterium Thiobacillus sp. W5

A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the V _max was 2.1 ± 0.1 μmol cytochrome c (mg protein)^–1 min^–1, and the K _m values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product. Received: 15 October 1996 / Accepted: 7 January 1997     

Thermal resistance parameters of the air environment at various altitudes

 The thermal properties of atmospheric air surrounding the human body at various altitudes are characterized with a system of parameters. This system comprises resistance of the air to convective heat transfer h _c ^–1, °C (W/m²)⁻¹ and to water vapour transfer h _D ^–1, s/m. The concept of ’evaporative resistance’h _e ^–1, hPa (W/m²)⁻¹) following the similarity of the processes is introduced. In obtaining the altitude dependencies of investigated paramters, a respective heat transfer equation expressing the rate of heat exchange at the boundary body surface – ambient air is applied. The use of the body thermal state of the established altitude dependencies is discussed. The concept of ’thermal stability’ related to the evaporative resistance parameter h _e ^–1 is introduced. This parameter is assumed as: (1) an indicator of the human body thermal stability and (2) distributor and predictor of environmental influence on the body thermal state. Received: 5 January 1996 / Accepted 5 November 1996     

Heat and mass exchange processes between the surface of the human body and ambient air at various altitudes

 The rates of convection and evaporation at the interface between the human body and the surrounding air are expressed by the parameters convective heat transfer coefficient h _c, in W m^–2°C^–1 and evaporative heat transfer coefficient h _e, W m^–2 hPa^–1. These parameters are determined by heat transfer equations, which also depend on the velocity of the airstream around the body, that is still air (free convection) and moving air (forced convection). The altitude dependence of the parameters is represented as an exponential function of the atmospheric pressure p: h _c∼p ⁿ and h _e∼p ^1–n, where n is the exponent in the heat transfer equation. The numerical values of n are related to airspeed: n=0.5 for free convection, n=0.618 when airspeed is below 2.0 ms^–1 and n=0.805 when airspeed is above 2.0 ms^–1. This study considers the coefficients h _c and h _e with respect to the similarity of the two processes, convection and evaporation. A framework to explain the basis of established relationships is proposed. It is shown that the thickness of the boundary layer over the body surface increases with altitude. As a medium of the transfer processes, the boundary layer is assumed to be a layer of still air with fixed insulation which causes a reduction in the intensity of heat and mass flux propagating from the human body surface to its surroundings. The degree of reduction is more significant at a higher altitude because of the greater thickness of the boundary layer there. The rate of convective and evaporative heat losses from the human body surface at various altitudes in otherwise identical conditions depends on the following factors: (1) during convection – the thickness of the boundary layer, plus the decrease in air density, (2) during evaporation (mass transfer) – the thickness of the boundary layer, plus the increase with altitude in the diffusion coefficient of water vapour in the air. The warming rate of the air volume due to convection and evaporation is also considered. Expressions for the calculation of altitude dependences h _c (p) and h _e (p) are suggested. Received: 23 June 1998 / Accepted: 10 February 1999