Cytochrome oxidation in bacterial photosynthesis

In this paper we propose that the reduction of the bacteriochlorophyl dimer cation (P⁺) by cytochrome c in the photosynthetic bacteria Rps. viridis and Chromatium vinosum proceeds via two parallel electron transfer (ET) processes from two distinct cytochrome c molecules. The dominating ET process at high temperatures involves the activated oxidation of the high-potential cytochrome c at closest proximity to P, while the dominating low-temperature process involves activationless ET from a low-potential cytochrome c, which is further away from P. The available data for the effects of blocking the low-potential cytochrome c on ET dynamics are consistent with this model, which results in reasonable nuclear reorganization and electronic coupling parameters for the parallel cytochrome oxidation processes. The lack of universality in the cytochrome oxidation in reaction centres of various bacteria is emphasized.     

The role of a cytochrome c-552-cytochrome c complex in the oxidation of sulfide in Chromatium vinosum

The sulfide:cytochrome c oxidoreductase activity of the flavocytochrome c-522 from the purple sulfur bacterium Chromatium vinosum has been investigated. The oxidized sulfur product of the sulfide:cytochrome c reductase activity has been shown to be elemental sulfur. Cytochrome c-552 has been found to form a stable complex with horse heart cytochrome c that appears to be held together by electrostatic interactions. The stability of this complex and the sulfide:cytochrome c reductase activity of cytochrome c-552 are both ionic strength dependent, with maximal rates of cytochrome c reduction and extent of complex formation occurring over the same ionic strength range. Trifluoroacetylated cytochrome c is not reduced in the presence of cytochrome c-552 and sulfide, nor does it form a complex with cytochrome c-552. These results suggest the possible involvement of cytochrome c lysine residues in complex formation. Cytochrome c-552 migrates with an anomalously high apparent molecular weight on gel filtration columns equilibrated with low ionic strength buffers, suggesting the possibility of conformational changes or dimerization of the protein. However, complexation of cytochrome c-552 with cytochrome c still occurs at low ionic strength.     

Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c₁ from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c₁, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c₁ is more exposed than is that of mammalian cytochrome c₁, but less exposed than that of cytochrome c₂. Ferricytochrome c₁ undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c₁, suggesting that the sulfhydryl groups of cytochrome c₁ are the electron donors for photoreduction. Purified cytochrome c₁ contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c₁, the bacterial protein does not form a stable complex with cytochrome c₂ or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c₁ and bacterial ferricytochrome c₂, and between bacterial ferrocytochrome c₁ and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c₁ is calculated to be 228 mV, which is identical to that of mammalian cytochrome c₁.     

The role of protein dynamics and thermal fluctuations in regulating cytochrome c/cytochrome c oxidase electron transfer

In this overview we present recent combined electrochemical, spectroelectrochemical, spectroscopic and computational studies from our group on the electron transfer reactions of cytochrome c and of the primary electron acceptor of cytochrome c oxidase, the Cu_A site, in biomimetic complexes. Based on these results, we discuss how protein dynamics and thermal fluctuations may impact on protein ET reactions, comment on the possible physiological relevance of these results, and finally propose a regulatory mechanism that may operate in the Cyt/CcO electron transfer reaction in vivo. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.     

Different temperature dependencies of the charge recombination reaction in photoreaction centers isolated from different bacterial species

We compared the temperature dependency of the rate of the charge recombination reaction in photoreaction centers isolated from Ectothiorhodospira sp. and from Rhodospirillum rubrum G9. We also examined the temperature dependency of the bandwidth and peak wavelength of their far-red absorption band. In both preparations, the peak wavelength and the bandwidth vary monotonically with temperature between 80 and 300 K. However, the rate of the charge recombination reaction has a quite different temperature dependency. In the preparation from R. rubrum, the reaction is accelerated 5-fold in a typical sigmoidal fashion as the temperature is lowered from 300 to 80 K. In the preparation from Ectothiorhodospira sp., the reaction is accelerated monotonically only about 1.5-fold in the same temperature range. At temperatures below 100 K, the rates are similar in the two preparations. We interpret the temperature dependency of the charge recombination reaction in terms of an activationless electron-transfer model formulated by Jortner (Jortner, J. (1980) Biochim. Biophys. Acta 394, 193–230). The minimal model provides a good fit for the temperature dependency of charge recombination in the preparation from Ectothiorhodospira sp. However, to fit the temperature dependency of the R. rubrum preparation with the same model, we must further postulate that the electronic coupling factor varies with temperature in this preparation. We find that, in both preparations, the temperature dependency of the far-red absorption bandwidth is consistent with the assumption that similar vibrational modes are involved in electron transfer and in electronic excitation.     

Cytochrome c1 and b-type cytochrome with two heme centers in the photosynthetic membranes from Rhodopseudomonas palustris

The photosynthetic membranes from Rhodopseudomonas palustris contained one species of membrane-bound c-type cytochrome, presumably cytochrome c₁, and a b-type cytochrome with two heme centers. The molecular weight and midpoint potential of cytochrome c₁ were 30000 and 275 mV, respectively. The peak of the reduced-minus-oxidized difference spectrum of cytochrome c₁ was at 552 nm. Molecular weight of the b-type cytochrome was 32000 and the cytochrome had two midpoint potentials of 60 mV and −55 mV. The peaks of the reduced-minus-oxidized difference spectra of the high and low midpoint potential heme centers were at 560 and 562 nm, respectively. These results suggested that there was a cytochrome b-c₁ complex in Rps. palustris.     

Dielectric constant dependence of biological oxidation-reduction. 1. A model of polarity-dependent ferrocytochrome c oxidation

A theoretical model for the effect of the dielectric constant (c) of the solvent medium on ferrocytochrome c oxidation by ferricyanide is developed to account for the observed variations of the rate constant (k) of reactions in aqueous binary mixtures with alcohols (less than 5-10 mol% ethanol and propranolol). A correlation between k and c is found if ln k is expressed as a function of the Kirkwood parameter (c-1)(2c+1). The results of calculations indicate that the use of the 'overall dipole moment' of cytochrome c in oxidoreduction studies is likely to be unreliable. Instead, the decrease in k in alcohol/water mixtures is best explained--in conformity with Onsager's theory of the reaction field--by a polarity effect on the dipole moment of the cytochrome c heme upon diffusion of the polar solvent molecules into the low dielectric constant heme crevice.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH₃CH(OH), CO₂, O₂, and e⁻_aq′ and the oxidation of the reduced cytochrome c derivatives by Fe(CN)³⁻₆ were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partitioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

On the mechanism of magnetic field effects in bacterial photosynthesis.

The recently discovered magnetic field effects in bacterial photosynthesis are discussed by solving a simple model exactly. Analytic expressions are given that permit one to study the influence of the rates of the primary electron transfer reactions and the exchange interaction on the yield of excited triplet states.     

Electron transfer reactions of cytochrome c confined within erythrocyte ghosts

We have prepared and characterized resealed erythrocyte ghosts in which the only discernible pigment is cytochrome c. The resealed ghosts have the normal orientation and are free of ‘leaky’ species; they are stable and can be maintained at 4°C for many days without lysis.

The internal cytochrome c participates in redox reactions with both soluble and insolubilized cytochrome c present externally, and with external cytochrome b₅. No reaction was observed with plastocyanin, cytochrome c oxidase or NADPH-cytochrome c reductase.

A study has been made of the reaction of the internal cytochrome c with the low molecular weight reductants, ascorbate and glutathione. Complex kinetics are observed with both reagents: with ascorbate the results are best explained by assuming the existence, in the membrane, of a redox-active species able to undergo dedimerization. A protein bound disulfide bond would satisfy the requirement.     

Electrochemical approach to the mechanism of urea denaturation of horse heart cytochrome c

The effect of urea denaturation on the electroactivity of horse heart cytochrome c has been studied by differential pulse polarography and cyclic voltammetry at a gold electrode; the gold electrode was activated by 4,4'-bipyridine. Essentially, two redox couples with E₀₁ ≈ 0.25 V and E'_oz ≈ ?0.05 V (vs. normal hydrogen electrode) have been detected. The experimental results have been interpreted on the basis of the existence of equilibria between native and denatured electroactive forms; transitory species have been assumed to appear on reduction. The scheme that we have proposed agrees well with the conclusions obtained previously by other authors on conformational changes. Moreover, the advantage of electrochemical techniques in investigating the denaturation process has been underlined.     

Location of a cytochrome c binding site on the surface of flavocytochrome b 2

Saccharomyces cerevisiae flavocytochrome b ₂ couples the oxidation of L-lactate to the reduction of cytochrome c. The second-order rate constant for cytochrome c reduction by flavocytochrome b ₂ depends on the rate of complex formation and is sensitive to ionic strength. Mutations in the heme domain of flavocytochrome b ₂ (Glu63→Lys, Asp72→Lys and the double mutation Glu63→Lys:Asp72→Lys) have significant effects on the reaction with cytochrome c, implicating these residues in complex formation. This kinetic information has been used to guide molecular modelling studies, which are consistent with there being no one single best-configuration. Rather, there is a set of possible complexes in which the docking-face of cytochrome c can approach flavocytochrome b ₂ in a variety of orientations. Four cytochromes c can be accommodated on the flavocytochrome b ₂ tetramer, with each cytochrome c forming interactions with only one flavocytochrome b ₂ subunit. All the models involve residues 72 and 63 on flavocytochrome b ₂ but in addition predict that Glu237 may also be important for complex formation. These acidic residues interact with the basic residues 13, 27 and 79 on cytochrome c. Through this triangle of interactions runs a possible σ-tunnelling pathway for electron transfer. This pathway starts with the imidazole ring of His66 (a ligand to the heme-iron of flavocytochrome b ₂) and ends with the ring of Pro68, which is in van der Waals contact with the cytochrome c heme. In total, the edge-to-edge "through space" distance from the imidazole ring of His66 to the C3C pyrrole ring of cytochrome c is 13.1?Å.     

Evidence for an anisotropic magnetic interaction between the (bacteriopheophytin) intermediary acceptor and the first quinone acceptor in bacterial photosynthesis

We have investigated electron spin polarization effects occurring in protonated and perdeuterated reaction centers of Rhodospirillum rubrum with electron spin resonance at 9 and 35 GHz (X- and Q-band). As for Rhodopseudomonas sphaeroides strains 2.4.1 and R-26 (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 520–535; Gast, P., Mushlin, R.A. and Hoff, A.J. (1982) J. Phys. Chem. 86, 2886–2891), electron spin polarization effects of the prereduced first quinone acceptor Q^?_A in R. rubrum are strongly nonuniform. This nonuniformity is due to an anisotropic magnetic coupling between the intermediary bacteriopheophytin acceptor (I^?) and Q^?_A. It is argued that the anisotropy is too strong to arise solely from an anisotropy in the exchange interaction between I^? and Q^?_A and that dipolar contributions to the magnetic coupling between I^? and Q^?_A are important. The anisotropy in the magnetic coupling for reaction centers of Rps. sphaeroides strains 2.4.1 and R-26 is different from that of R. rubrum wild type. The combination of the 4-fold higher resolution at Q-band and the line narrowing upon deuteration has enabled us to obtain the principal g values and two hyperfine interaction constants of the reduced first quinone acceptor Q^?_A. The principal g values are g_x = 2.0067, g_y = 2.0056 and g_z = 2.0024; the hyperfine constant of the CH₂ group at position 1 is 1.6 G and that of the CH₃ group at position 2 is 2.1 G. These values are close to those found for ubisemiquinone in vitro (Okamura, M.Y., Debus, R.J., Isaacson, R.A. and Feher, G. (1980) Fed. Proc. 39, 1802; Hales, B.J. (1975) J. Am. Chem. Soc. 97, 5993–5997).     

Redox-Bohr effect in electron/proton energy transduction: cytochrome c 3 coupled to hydrogenase works as a 'proton thruster' in Desulfovibrio vulgaris

 A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c ₃, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement. Received: 2 April 1997 / Accepted: 23 May 1997     

Electronic interactions between iron and the bound semiquinones in bacterial photosynthesis. EPR spectroscopy of oriented cells of Rhodopseudomonas viridis

Electron paramagnetic resonance (EPR) spectroscopy of the iron-semiquinone complex in photosynthetic bacterial cells and chromatophores of Rhodopseudomonas viridis is reported. Magnetic fields are used to orient the prolate ellipsoidal-shaped cells which possess a highly ordered internal structure, consisting of concentric, nearly cylindrical membranes. The field-oriented suspension of cells exhibits a highly dichroic EPR signal for the iron-semiquinone complex, showing that the iron possesses a low-symmetry ligand field and exists in a preferred orientation within the native reaction-center membrane complex. The EPR spectrum is analyzed utilizing a spin hamiltonian formalism to extract physical information describing the electronic structure of the iron and the nature of its interaction with the semiquinones. Exact numerical solutions and analytical expressions for the transition frequencies and intensities derived from a perturbation theory expansion are presented, and a computer-simulated spectrum is given. It has been found that, for a model which assumes no preferred orientation within the plane of the membranes, the orientation of the Fe²⁺ ligand axis of largest zero-field splitting (Z, the principal magnetic axis) is titled 64±6° from the membrane normal. The ligand field for Fe²⁺ has low symmetry, with zero-field splitting parameters of |D₁|=7.0±1.3 cm^?1 and |E₁|=1.7±0.5 cm^?1 and $\text{|}\text{E}{}_1\text{D}{}_1\text{|=0.26}$ for the redox state Q₁^?Fe²⁺Q^2?. The rhombic character of the ligand field is increased in the redox state Q₁Fe²⁺Q^?₂, where $\text{0.33>|}\text{E}{}_2\text{D}{}_2\text{|>0.26}$. This indicates that the redox state of the quinones can influence the ligand field symmetry and splitting of the Fe²⁺. There exists an electron-spin exchange interaction between Fe²⁺ and Q^?₁ and Q^?₂, having magnitudes |J₁|=0.12±0.03 cm^?1 and $\text{|J}{}_2\text{|?0.06}\text{cm}{}^{\mathrm{?1}}$, respectively. Such weak interactions indicate that a proper electronic picture of the complex is as a pair of immobilized semiquinone radicals having very little orbital overlap (probably fostered by superexchange) with the Fe²⁺ orbitals. The exchange interaction is analyzed by comparison with model systems of paramagnetic metals and free radicals to indicate an absence of direct coordination between Fe²⁺ and Q^?₁ and Q^?₂. Selective line-broadening of some of the EPR transitions, involving Q^? coupling to the magnetic sublevels of the Fe²⁺ ground state, is interpreted as arising from an electron-electron dipolar interaction. Analysis of this line-broadening indicates a distance of 6.2–7.8 ? between Fe²⁺ and Q^?₁, thus placing Q₁ outside the immediate coordination shell of Fe²⁺.     

Quantum entanglement in photoactive prebiotic systems

This paper contains the review of quantum entanglement investigations in living systems, and in the quantum mechanically modelled photoactive prebiotic kernel systems. We define our modelled self-assembled supramolecular photoactive centres, composed of one or more sensitizer molecules, precursors of fatty acids and a number of water molecules, as a photoactive prebiotic kernel systems. We propose that life first emerged in the form of such minimal photoactive prebiotic kernel systems and later in the process of evolution these photoactive prebiotic kernel systems would have produced fatty acids and covered themselves with fatty acid envelopes to become the minimal cells of the Fatty Acid World. Specifically, we model self-assembling of photoactive prebiotic systems with observed quantum entanglement phenomena. We address the idea that quantum entanglement was important in the first stages of origins of life and evolution of the biospheres because simultaneously excite two prebiotic kernels in the system by appearance of two additional quantum entangled excited states, leading to faster growth and self-replication of minimal living cells. The quantum mechanically modelled possibility of synthesizing artificial self-reproducing quantum entangled prebiotic kernel systems and minimal cells also impacts the possibility of the most probable path of emergence of protocells on the Earth or elsewhere. We also examine the quantum entangled logic gates discovered in the modelled systems composed of two prebiotic kernels. Such logic gates may have application in the destruction of cancer cells or becoming building blocks of new forms of artificial cells including magnetically active ones.     

Thermodynamic and kinetic properties of the outer membrane cytochrome OmcF,a key protein for extracellular electron transfer in Geobacter sulfurreducens

Gene knock-out studies on Geobacter sulfurreducens have shown that the monoheme c-type cytochrome OmcF is essential for the extracellular electron transfer pathways involved in the reduction of iron and uranium oxy-hydroxides, as well as, on electricity production in microbial fuel cells. A detailed electrochemical characterization of OmcF was performed for the first time, allowing attaining kinetics and thermodynamic data. The heterogeneous electron transfer rate constant was determined at pH?7 (0.16?±?0.01?cm?s^?1) indicating that the protein displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The analysis of the structures of OmcF allowed us to assign the redox-Bohr centre to the side chain of His⁴⁷ residue and its pK_a values in the reduced and oxidized states were determined (pK_ox?=?6.73; pK_red?=?7.55). The enthalpy, entropy and Gibbs free energy associated with the redox transaction were calculated, pointing the reduced form of the cytochrome as the most favourable. The data obtained indicate that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF.     

Redox-Bohr effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms

 Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c ₃ from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e^– step, ideally suited for the coupling role of cytochrome c ₃ to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H₂ as energy source, cytochrome c ₃ can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.