Potential regulates metabolism and extracellular respiration of electroactive Geobacter biofilm

Dissimilatory metal reducer Geobacter sulfurreducens can mediate redox processes through extracellular electron transfer and exhibit potential-dependent electrochemical activity in biofilm. Understanding the microbial acclimation to potential is of critical importance for developing robust electrochemically active biofilms and facilitating their environmental, geochemical, and energy applications. In this study, the metabolism and redox conduction behaviors of G. sulfurreducens biofilms developed at different potentials were explored. We found that electrochemical acclimation occurred at the initial hours of polarizing G. sulfurreducens cells to the potentials. Two mechanisms of acclimation were found, depending on the polarizing potential. In the mature biofilms, a low level of biosynthesis and a high level of catabolism were maintained at +0.2 V versus standard hydrogen electrode (SHE). The opposite results were observed at potentials higher than or equal to +0.4 V versus SHE. The potential also regulated the constitution of the electron transfer network by synthesizing more extracellular cytochrome c such as OmcS at 0.0 and +0.2 V and exhibited a better conductivity. These findings provide reasonable explanations for the mechanism governing the electrochemical respiration and activity in G. sulfurreducens biofilms.     

Redox potentials of trinuclear μ-oxo ruthenium acetate clusters with N-heterocyclic ligands

The trinuclear clusters of general composition [Ru₃O(OOCCH₃)₆(N-Het)₃], where N-Het=pyridine and pyrazine derivatives, exhibit a series of reversible waves in the range of −1.8 to 2.4 V versus SHE, in acetonitrile, ascribed to the successive [cluster]^{−2/−1/0/+1/+2/+3} redox couples. The redox potentials decrease with the pK_a of the N-heterocyclic ligands according to the equations E°(+3/+2)= 2.24−0.023 pK_a; E°(+2/+1)=1.34−0.029 pK_a; E°(+1/0)=0.36−0.039 pK_a and E°(0/−1)=−0.68− 0.074 pK_a. The dependence is greater at lower oxidation states, reflecting the role of π-backbonding in the complexes.     

The impact of electron donors and anode potentials on the anode-respiring bacteria community

Both anode potentials and substrates can affect the process of biofilm formation in bioelectrochemical systems, but it is unclear who primarily determine the anode-respiring bacteria (ARB) community structure and composition. To address this issue, we divided microbial electrolysis cells (MECs) into groups, feeding them with different substrates and culturing them at various potentials. Non-turnover cyclic voltammetry indicated that the extracellular electron transfer components were uniform when feeding acetate, because the same oxidation peaks occurred at − 0.36 ± 0.01 and − 0.17 ± 0.01 V (vs. Ag/AgCl). Illumina MiSeq sequencing revealed that the dominating ARB was Geobacter, which did not change with different potentials. When the MECs were cultured with sucrose and mixed substrates, oxidation peak P3 (− 0.29 ± 0.015 V) occurred at potentials of − 0.29 and 0.01 V. This may be because of the appearance of Unclassified_AKYG597. In addition, oxidation peak P4 (− 0.99 ± 0.01 V) occurred at high and low potentials (0.61 and − 0.45 V, respectively), and the maximum current densities were far below those of the middle potentials. Illumina MiSeq sequencing showed that fermentation microorganisms (Lactococcus and Sphaerochaeta) dominated the biofilms. Consequently, substrate primarily determined the dominating ARB, and Geobacter invariably dominated the acetate-fed biofilms with potentials changed. Conversely, different potentials mainly affected fermentable substrate-fed biofilms, with dominating ARB turning into Unclassified_AKYG59.

     

pH, redox potential and local biofilm potential microenvironments within Geobacter sulfurreducens biofilms and their roles in electron transfer

The limitation of pH inside electrode‐respiring biofilms is a well‐known concept. However, little is known about how pH and redox potential are affected by increasing current inside biofilms respiring on electrodes. Quantifying the variations in pH and redox potential with increasing current is needed to determine how electron transfer is tied to proton transfer within the biofilm. In this research, we quantified pH and redox potential variations in electrode‐respiring Geobacter sulfurreducens biofilms as a function of respiration rates, measured as current. We also characterized pH and redox potential at the counter electrode. We concluded that (1) pH continued to decrease in the biofilm through different growth phases, showing that the pH is not always a limiting factor in a biofilm and (2) decreasing pH and increasing redox potential at the biofilm electrode were associated only with the biofilm, demonstrating that G. sulfurreducens biofilms respire in a unique internal environment. Redox potential inside the biofilm was also compared to the local biofilm potential measured by a graphite microelectrode, where the tip of the microelectrode was allowed to acclimatize inside the biofilm. Biotechnol. Bioeng. 2012; 109: 2651–2662. © 2012 Wiley Periodicals, Inc.     

Production of bioelectricity,bio-hydrogen,high value chemicals and bioinspired nanomaterials by electrochemically active biofilms

Microorganisms naturally form biofilms on solid surfaces for their mutual benefits including protection from environmental stresses caused by contaminants, nutritional depletion or imbalances. The biofilms are normally dangerous to human health due to their inherited robustness. On the other hand, a recent study suggested that electrochemically active biofilms (EABs) generated by electrically active microorganisms have properties that can be used to catalyze or control the electrochemical reactions in a range of fields, such as bioenergy production, bioremediation, chemical/biological synthesis, bio-corrosion mitigation and biosensor development. EABs have attracted considerable attraction in bioelectrochemical systems (BESs), such as microbial fuel cells and microbial electrolysis cells, where they act as living bioanode or biocathode catalysts. Recently, it was reported that EABs can be used to synthesize metal nanoparticles and metal nanocomposites. The EAB-mediated synthesis of metal and metal–semiconductor nanocomposites is expected to provide a new avenue for the greener synthesis of nanomaterials with high efficiency and speed than other synthetic methods. This review covers the general introduction of EABs, as well as the applications of EABs in BESs, and the production of bio-hydrogen, high value chemicals and bio-inspired nanomaterials.     

Electron redistribution in mixed valence cytochrome oxidase following photolysis of carboxy-oxidase

Absorbance changes at 446 nm in purified cytochrome oxidase following flash photolysis of carboxy-oxidase poised in the mixed valence state at +220 mV show biphasic kinetics. One phase corresponds to CO recombination to ferrous cytochromea ₃ with an energy of activation of 9 kcal/mol; the second phase is 3–5 times faster with an energy of activation of 9.15 kcal/mol. Following flash photolysis at approximately –60°C, cytochromesa andc and the 840-nm CuA species are observed to undergo reduction as electrons from ferrous unliganded cytochromea ₃ equilibrate with the equipotential redox centers of the oxidase; as CO recombines with ferrous cyochromea ₃, these centers are oxidized and the mixed valence carboxy-oxidase is regenerated. Electron redistribution between centers of the oxidase in the forward and reverse directions occurs faster than does the binding of CO.     

Electrostatics of the FeS clusters in respiratory complex I

Respiratory complex I couples the transfer of electrons from NADH to ubiquinone and the translocation of protons across the mitochondrial membrane. A detailed understanding of the midpoint reduction potentials (E_m) of each redox center and the factors which influence those potentials are critical in the elucidation of the mechanism of electron transfer in this enzyme. We present accurate electrostatic interaction energies for the iron-sulfur (FeS) clusters of complex I to facilitate the development of models and the interpretation of experiments in connection to electron transfer (ET) in this enzyme. To calculate redox titration curves for the FeS clusters it is necessary to include interactions between clusters, which in turn can be used to refine E_m values and validate spectroscopic assignments of each cluster. Calculated titration curves for clusters N4, N5, and N6a are discussed. Furthermore, we present some initial findings on the electrostatics of the redox centers of complex I under the influence of externally applied membrane potentials. A means of determining the location of the FeS cofactors within the holo-complex based on electrostatic arguments is proposed. A simple electrostatic model of the protein/membrane system is examined to illustrate the viability of our hypothesis.     

Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a₃ (Fe_a3) and copper (Cu_B). Here, the influence of the ligation at the oxidized Fe_a3³⁺–Cu_B²⁺ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (E_m(a)) for the cyanide (the low-spin Fe_a3³⁺) and the formate-ligated CcO (the high-spin Fe_a3³⁺). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of E_m(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fe_a³⁺ between His–Fe_a³⁺–His and His–Fe_a³⁺–OH⁻ occurs only at pH above 9.5 with the estimated pK >11.0.     

Microbiological Redox Potential Control to Improve the Efficiency of Chalcopyrite Bioleaching

The effect of controlling the redox potential (E_h) on chalcopyrite bioleaching kinetics was studied as a new aspect of redox control during chalcopyrite bioleaching, and its mechanism was investigated by employing the “normalized” solution redox potential (E_normal) and the reaction kinetics model. Different E_h ranges were established by use of different acidophiles (Sulfobacillus acidophilus YTF1; Sulfobacillus sibiricus N1; Acidimicrobium ferrooxidans ICP; Acidiplasma sp. Fv-AP). Cu dissolution was very susceptible to real-time change in E_h during the reaction. It was found that efficiency of bioleaching of chalcopyrite can be effectively evaluated on the basis of E_normal, since it is normalized for real-time fluctuations of concentrations of major metal solutes during bioleaching. For steady Cu solubilization during bioleaching at a maximum rate, it was important to maintain a redox potential range of 0 ≤ E_normal ≤ 1 (?0.35 mV optimal) at the mineral surface by employing a “weak” ion-oxidizer. This led to a copper recovery of > 75%. At higher E_normal levels (E_normal > 1 by “strong” microbial Fe²⁺ oxidation), Cu solubilization was slowed by diffusion through the product film at the mineral surface (< 50% Cu recovery) caused by low reactivity of the chalcopyrite and by secondary passivation of the chalcopyrite surface, mainly by jarosite.     

Fluorescence decline as a function of redox potential and actinic light intensity in spinach thylakoids

Excitation of isolated thylakoids with sufficiently strong actinic light increases the fluorescence quantum yield up to a maximum level, F_max, followed by a slower decline under certain experimental conditions. In this study the latter effect was analyzed as a function of the ambient redox potential and the actinic light intensity. Two different types of fluorescence decrease were found. (a) In the presence of specific quinones widely used as redox mediators a fast and comparatively small decrease (30% of F_max), referred to as ΔF_SQ, was observed at moderate redox potentials (−300 <E_m < + 200 mV). ΔF_SQ disappears at positive values with E_{m, 7.5} = + 110 mV, whereas the decrease at negative redox potential depends on the midpoint potential of the quinone. (b) A more pronounced fluorescence decline was observed at redox potentials below −300 mV, which comprises 65–70% of the maximum fluorescence. The full expression of this effect, referred to as ΔF^max_LP, requires markedly higher actinic light intensities than ΔF^max_SQ. The extent of ΔF^max_LP as a function of the redox potential is dependent on the presence of redox mediators. In their absence the full expression of ΔF^max_LP can be only observed below −400 mV. Based on the hypothesis of Pheo⁻ photoaccumulation being responsible for the fluorescence decline at low redox potentials (Klimov, V.V., Klevanik, A.V. and Shuvalov, V.A. (1977) FEBS Lett. 82, 182–186), a reaction scheme is presented that qualitatively describes the time course of ΔF_LP at different actinic light intensities and redox potentials. Based on this analysis, the rate of Pheo^⨪ reoxidation is inferred to be limited by the reaction center apoprotein acting as a barrier to redox equilibration. The implications for the interpretations of redox titration curves are briefly discussed.     

Metabolic Efficiency of Geobacter sulfurreducens Growing on Anodes with Different Redox Potentials

Microorganisms respiring Fe(III) in the environment face a range of redox potentials of the prospective terminal ferric electron acceptors, because Fe(III) can be present in different minerals or organic complexes. We investigated the adaptation of Geobacter sulfurreducens to this range by exposing the bacteria to different redox potentials between the electron donor acetate and solid, extracellular anodes in a microbial fuel-cell set-up. Over a range of anode potentials from ?0.105 to +0.645 V versus standard hydrogen electrode, G. sulfurreducens produced identical amounts of biomass per electron respired. This indicated that the organism cannot utilize higher available energies for energy conservation to ATP, and confirmed recent studies. Either the high potentials cannot be used due to physiological limitations, or G. sulfurreducens decreased its metabolic efficiency, and less biomass per unit of energy was produced. In this case, G. sulfurreducens “wasted” energy at high-potential differences, most likely as heat to fuel growth kinetics.     

Adaptations of tropical marine microphytobenthic assemblages along a gradient of light and nutrient availability in Suva Lagoon,Fiji

How are microphytobenthic biofilms adapted to the high incident irradiances and temperatures, low inorganic nutrient concentrations and high desiccation stresses on intertidal flats present in tropical environments? This study investigated biofilms subject to different environmental conditions in a range of tropical sites in Suva lagoon, Fiji. PAM fluorescence was used to measure photophysiological responses to the light climate. Biofilm colloidal carbohydrate, extracellular polymeric substances (EPS) and low molecular weight (MW) carbohydrate concentrations and diel carbohydrate production patterns were measured. Average biomass (Chl a) ranged from 15 to 36?mg?m^?2, and was highest in seagrass bed sediments, but biomass was not correlated with water column or sediment porewater nutrient concentrations. Biofilm photophysiology differed significantly along a combined gradient of light and nutrient availability, with F _v/F _m, relative ETR_max and E _k of biofilms highest in mangrove and intertidal main island sites and lowest in subtidal coral reef flats. Subtidal biofilms showed photoinhibition at irradiances > 1000?µmol?m^?2. Significant correlations between Chl a and colloidal carbohydrate concentrations were present (except on intertidal sandflats), and tropical biofilms had higher ratios of colloidal carbohydrate and EPS to Chl a than temperate estuarine biofilms, probably due to a combination of high irradiance and low nutrient availability leading to the production of excess photoassimilates. The percentage of EPS present in the colloidal fraction was highest in coral sand biofilms (42%), which had the lowest nutrient concentrations, compared with other sites (25–32%). Intertidal biofilms predominantly consisted of large motile taxa and showed strong rhythms of vertical migration. During tidal emersion, high sediment temperatures (41?°C), irradiance (>2300?µmol?m^?2?s^?1) and salinity (49‰) stimulated downward migration. In silty sediments, migration resulted in a reduction in photosynthetic activity during the midday period but, in sands with high light penetration (to a depth of > 1700?µm), high production rates of EPS (18.2?µg carbo. µg Chl a^?1 h^?1) and low MW carbohydrate exudates (40.2?µg carbo. µg Chl a^?1 h^?1) occurred. Vertical migration, high E _k and high rates of photoassimilate dumping are all adaptations to living in the tropical intertidal zone. Seagrass and reef flat biofilms consisted of a diverse non-migratory flora of motile and non-motile taxa that were not subject to such extreme temperature and irradiance conditions. Low values of photosynthetic parameters and high colloidal and EPS content indicated that these biofilms were nutrient-limited.     

Surface coordination chemistry of noble-metal electrocatalysts: Oxidative addition and reductive elimination of iodide at iridium,platinum and gold in aqueous solutions

The oxidative addition and reductive elimination of the iodo ligand has been compared at smooth polycrystalline gold, platinum and iridium surfaces in aqueous solutions. On these three metals, the iodo species undergoes spontaneous oxidative chemisorption to form a close-packed monolayer of zero-valent iodine, the saturation coverage of which is limited by the van der Waals radius of the iodine atom; this oxidative addition process is further manifested by evolution of hydrogen gas from proton reduction. Elimination of iodine from these surfaces can be achieved by its reduction back to the anion either by application of sufficiently negative potentials or by exposure to ample amounts of hydrogen gas. On Pt and Ir, the reductive desorption of iodine is coupled with reductive chemisorption of hydrogen; consequently, the reaction is a two-electron, pH-dependent process. A plot of E_1/2, the potential at which the iodine coverage is decreased to half its maximum value, against pH yields information concerning the redox potential of the I_(ads)/I⁻_(ads) couple in the surface-coordinated state. On Au, where dissociative chemisorption of hydrogen does not occur, the iodine-stripping process is a pH-independent, one-electron reaction. The difference in the redox potentials [E^o_I(ads) -E^o_I(aq] for the I_(ads) and I_2(aq)/I⁻_(aq) redox couples was found to be −0.90 V on Au, − 0.76 V on Pt, and −0.72 V on Ir. These values imply that the ratio of the formation constants for surface coordination of the iodine and iodide species (K_f,I/K_f,I−) is 2 × 10²⁸ on Au, 1 × 10²⁶ on Pt, and 2 × 10²⁵ on Ir.     

Electrophysiology of phagocytic membranes: intracellular K+ activity and K+ equilibrium potential in macrophage polykaryons

The role of K⁺ as current carrier during the slow membrane hyperpolarizations (SH) elicited by iontophoretic Ca²⁺ injections into macrophage polykaryons is studied. The intracellular K⁺ activity (a_K) and the K⁺ equilibrium potential (E_K) are measured using ion-sensitive microelectrodes. The mean value of a_K is 84 ± 5 mM in a culture medium containing 5.3 mM K⁺, but increases to 100 ± 8 mM when the extracellular K⁺ concentration is raised to 30.3 mM. Under the same conditions the values of E_K obtained from the Nernst equation are −81 ± 2 mV and −40 ± 2 mV, respectively. The reversal potentials (E_R) of the SH are calculated from changes observed in transmembrane potential and input resistance, according to an equivalent model based only on passive ionic fluxes. The mean E_R values obtained are −74 ± 8 mV in the presence of low K⁺ concentration and −37 ± 3 mV for the high K⁺ medium. These values are significantly smaller than the estimated E_K for the corresponding situations. Evidence for the existence of an electrogenic (Na⁺ + K⁺)-ATPase activity is also presented. The evidence indicates that an increase in the membrane potassium permeability can account for about 90% of the total permeability change occurring during the SH.     

Comparative bioelectrochemical analysis of Pseudomonas aeruginosa and Escherichia coli with anaerobic consortia as anodic biocatalyst for biofuel cell application

Aims: To study the bioelectrochemical behaviour of Pseudomonas aeruginosa (MTCC 17702) and Escherichia coli (MTCC 10436) and to assess their potential to act as anodic biocatalyst with the function of anaerobic consortia for microbial (bio) fuel cell (BFC) application. Methods and Results: Three BFCs (single chamber; open‐air cathode; noncatalysed electrodes) were operated simultaneously in acidophilic microenvironments. Pseudomonas aeruginosa (BFC_P) showed higher current density (264 mA m^?2) followed by mixed culture (BFC_M; 166 mA m^?2) and E. coli (BFC_E; 147 mA m^?2). However, total operating period and substrate degradation were relatively found to be effective with mixed culture (58%; 72 h) followed by BFC_P (39%; 60 h) and BFC_E (31%; 48 h). Higher electron discharge (ED) was observed with Ps. aeruginosa while mixed culture showed the involvement of redox mediators in the ED process. Conclusions: Mixed culture showed to sustain biopotential for longer periods along with a stable ED. The presence of redox signals and high substrate degradation was also evidencing its performance compared to the pure strains studied. This supports the practical utility of mixed culture over the pure cultures for real‐field BFC applications especially while operating with wastewater. Significance and Impact of the Study: This study revealed the efficiency and viability of mixed consortia in comparison with pure strains for microbial (bio) fuel cell applications.     

Comparative redox and pK a calculations on cytochrome c 3 from several Desulfovibrio species using continuum electrostatic methods

 A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c ₃ family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pK _a calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c ₃ redox potentials in all the c ₃ molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c ₃ family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c ₃ from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely. Received: 31 August 1998 / Accepted: 20 November 1998     

Photosystem II heterogeneity: the acceptor side

It is well known that two photosystems, I and II, are needed to transfer electrons from H₂O to NADP⁺ in oxygenic photosynthesis. Each photosystem consists of several components: (a) the light-harvesting antenna (L-HA) system, (b) the reaction center (RC) complex, and (c) the polypeptides and other co-factors involved in electron and proton transport. First, we present a mini review on the heterogeneity which has been identified with the electron acceptor side of Photosystem II (PS II) including (a) L-HA system: the PS II and PS II units, (b) RC complex containing electron acceptor Q₁ or Q₂; and (c) electron acceptor complex: Q_A (having two different redox potentials Q_L and Q_H) and Q_B (Q_B-type; Q'_B type; and non-Q_B type); additional components such as iron (Q-400), U (E_m,7=–450 mV) and Q-318 (or Aq) are also mentioned. Furthermore, we summarize the current ideas on the so-called inactive (those that transfer electrons to the plastoquinone pool rather slowly) and active reaction centers. Second, we discuss the bearing of the first section on the ratio of the PS II reaction center (RC-II) and the PS I reaction center (RC-I). Third, we review recent results that relate the inactive and active RC-II, obtained by the use of quinones DMQ and DCBQ, with the fluorescence transient at room temperature and in heated spinach and soybean thylakoids. These data show that inactive RC-II can be easily monitored by the OID phase of fluorescence transient and that heating converts active into inactive centers.Abbreviations DCBQ 2,5 or 2,6 dichloro-p-benzoquinone - DMQ dimethylquinone - Q_A primary plastoquinone electron acceptor of photosystem II - Q_B secondary plastoquinone electron acceptor of photosystem II - IODP successive fluorescence levels during time course of chlorophyll a fluorescence: O for origin, I for inflection, D for dip or plateau, and P for peak     

Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing

Abstract

The half-cell reduction potential of the glutathione disulphide (GSSG)/glutathione (GSH) redox couple appears to correlate with cell viability and has been proposed to be a marker of seed viability and ageing. This study investigated the relationship between seed viability and the individual half-cell reduction potentials (E_is) of four low-molecular-weight (LMW) thiols in Lathyrus pratensis seeds subjected to artificial ageing: GSH, cysteine (Cys), cysteinyl-glycine (Cys-Gly) and γ-glutamyl-cysteine (γ-Glu-Cys). The standard redox potential of γ-Glu-Cys was previously unknown and was experimentally determined. The E_is were mathematically combined to define a LMW thiol-disulphide based redox environment (E_{thiol-disulphide}). Loss of seed viability correlated with a shift in E_{thiol-disulphide} towards more positive values, with a LD₅₀ value of ?0.90 ± 0.093 mV M (mean ± SD). The mathematical definition of E_{thiol-disulphide} is envisaged as a step towards the definition of the overall cellular redox environment, which will need to include all known redox-couples.     

A Periplasmic and Extracellular c-Type Cytochrome of Geobacter sulfurreducens Acts as a Ferric Iron Reductase and as an Electron Carrier to Other Acceptors or to Partner Bacteria

An extracellular electron carrier excreted into the growth medium by cells of Geobacter sulfurreducens was identified as a c-type cytochrome. The cytochrome was found to be distributed in about equal amounts in the membrane fraction, the periplasmic space, and the surrounding medium during all phases of growth with acetate plus fumarate. It was isolated from periplasmic preparations and purified to homogeneity by cation-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The electrophoretically homogeneous cytochrome had a molecular mass of 9.57 ± 0.02 kDa and exhibited in its reduced state absorption maxima at wavelengths of 552, 522, and 419 nm. The midpoint redox potential determined by redox titration was −0.167 V. With respect to molecular mass, redox properties, and molecular features, this cytochrome exhibited its highest similarity to the cytochromes c of Desulfovibrio salexigens and Desulfuromonas acetoxidans. The G. sulfurreducens cytochrome c reduced ferrihydrite (Fe(OH)₃), Fe(III) nitrilotriacetic acid, Fe(III) citrate, and manganese dioxide at high rates. Elemental sulfur, anthraquinone disulfonate, and humic acids were reduced more slowly. G. sulfurreducens reduced the cytochrome with acetate as an electron donor and oxidized it with fumarate. Wolinella succinogenes was able to reduce externally provided cytochrome c of G. sulfurreducens with molecular hydrogen or formate as an electron donor and oxidized it with fumarate or nitrate as an electron acceptor. A coculture could be established in which G. sulfurreducens reduced the cytochrome with acetate, and the reduced cytochrome was reoxidized by W. succinogenes in the presence of nitrate. We conclude that this cytochrome can act as iron(III) reductase for electron transfer to insoluble iron hydroxides or to sulfur, manganese dioxide, or other oxidized compounds, and it can transfer electrons to partner bacteria.     

A study of the kinetic properties of the stable semiquinone of the reaction-center secondary acceptor in chromatophores of non-sulfur purple bacteria

Flash-induced absorption changes at 450 nm were investigated in isolated chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum non-sulfur purple bacteria to follow the redox changes of the semiquinone species of the secondary quinone acceptor of the photosynthetic reaction center. Excitation of a dark-adapted chromatophore suspension by a series of successive flashes in the presence of electron donors capable of rapidly reducing the photooxidized reaction-center pigment causes the formation of a stable semiquinone species (Q^⨪_B) with a lifetime which is shown to be proportional to the amount of the oxidized redox mediator in the incubation medium. It is shown that the disappearance of the flash-induced absorption changes at 450 nm on lowering the ambient redox potential (E_h) to 200–300 mV is the result of increasing the lifetime of Q^⨪_B, as the amount of the oxidized mediator diminishes; consequently, in these circumstances, the 2–5 min dark interval between the flash cycles appears insufficient for Q^⨪_B recovery. After the addition of redox mediators with a low midpoint potential, acting as an oxidant for Q^⨪_B, the flash-induced redox changes of Q^⨪_B were observed at low E_h values unless E_h reached a value at which Q_B underwent reduction at equilibrium to form Q_BH₂. The data provide evidence that reaction centers with a fully oxidized secondary acceptor can donate electrons to the cyclic electron-transport chain only after two turnovers, leading to the formation of the doubly reduced ubiquinone species (Q_BH₂) of the secondary acceptor.