Alternative Cyanide-sensitive Oxidase Interacting with Photosynthesis in Synechocystis PCC6803. Ancestor of the Terminal Oxidase of Chlororespiration?

Influence of respiration on photosynthesis in Synechocystis PCC6803 was studied by measuring the redox transients of cytochrome f (cyt f) upon excitation of the cells with repetitive single turnover flashes. Upon the addition of KCN the flash-induced oxidation of cyt f was increased and the rereduction of cyt f+ was accelerated. Dependence of these effects on the concentration of KCN clearly demonstrated the existence of two cyanide-sensitive oxidases interacting with photosynthesis: cyt aa3, which was sensitive to low concentrations of cyanide, and an alternative oxidase, which could be suppressed by using 1 mM KCN. The interaction between the photosynthetic and the respiratory electron transport chains was regulated mainly by the activity of the alternative cyanide-sensitive oxidase. The oxidative pathway involving the alternative cyanide-sensitive oxidase was insensitive to salicyl hydroxamic acid and azide. The close resemblance of the inhibition pattern reported here and that described for chlororespiration in algae and higher plants strongly suggest that an oxidase of the same type as the alternative cyanide-sensitive oxidase of cyanobacteria functions as a terminal oxidase in chloroplasts.     

Oxygen reactions with bacterial oxidases and globins: binding,reduction and regulation

Oxygen is favoured as terminal electron acceptor in aerobic and facultative microorganisms because of its appropriate physical state, satisfactory solubility and its desirable combinations of kinetic and thermodynamic properties. Oxygen is generally reduced by four electrons to yield oxygen, but there are important biological consequences of, and roles for, the partial reduction to superoxide and peroxide. Complex and multiple regulatory networks ensure (i) the utilization of oxygen in preference to other oxidants, (ii) the synthesis of oxygen-consuming enzymes with appropriate properties (particularly affinity for the ligand), and (iii) appropriate cellular protection in the event of oxidative stress. This contribution reviews the terminal respiratory oxidases of selected Gram-negative bacteria and microbial haemoglobin-like proteins.Recent studies of the cytochromebd-type oxidases ofEscherichia coli andAzotobacter vinelandii suggest that, despite probable similarity at the amino acid level, the reactivities of these oxidases with oxygen are strikingly different. The respiratory protection afforded to nitrogenase in the obligately aerobic diazotrophA. vinelandii by the cytochromebd complex appears to be accompanied by, and may be the result of, a low affinity for oxygen and a high V_max. The poorly characterized cytochromeo-containing oxidase in this bacterium is not required for respiratory protection. InE. coli, the cytochromebd-type oxidase has a remarkably high affinity for oxygen, consistent with the view that this is an oxygen-scavenging oxidase utilized under microaerobic conditions. The demonstration of substrate (i.e. oxygen) inhibition in this complex suggests a mechanism whereby wasteful electron flux through a non-proton-pumping oxidase is avoided at higher dissolved oxygen tensions. The demonstration of two ligandbinding sites (haemsd andb ₅₉₅) in oxidases of this type suggests plausible mechanisms for this phenomenon. InE. coli, assembly of the cytochromebd-type oxidase (and of periplasmic cytochromesb andc) requires the presence of an ABC transporter, which may serve to export haem or some assembly factor' to the periplasm.There is at least one additional oxygen-consuming protein inE. coli — the flavohaemoglobin encoded by thehmp gene. Globin-like proteins are also widely distributed in other bacteria, fungi and protozoa, but most have unknown functions. The function of HMP and the related chimaeric flavohaemoglobins in other bacteria and yeast is unknown; one of several possibilities for HMP is that its relatively low affinity for oxygen during turnover with NADH as substrate could enable it to function as a sensor of falling (or rising) cytoplasmic oxygen concentrations.(until October 1994: Section of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA)     

A simple and fast method to analyze the orientation of c-type cytochromes in the outer membrane of Gram-negative bacteria

Outer membrane cytochromes catalyze the final reduction step of respiratory chains to electron acceptors that cannot diffuse through the outer membrane of Gram-negative bacteria. We developed an in vivo method to detect the orientation of outer membrane cytochromes via analysis of electron transfer reactions between these enzymes and riboflavin.     

Effect of hypoxia on the content and stoichiometry of cytochromes in muscle of the gray mullet Liza aurata

The maintenance of the gray mullet Liza aurata under conditions of hypoxia ( $ P_{O_2 } The maintenance of the gray mullet Liza aurata under conditions of hypoxia ( 55–82 GPa) for 1–2 days led to a decrease of oxygen tension in arterial blood by 47%, while in venous blood—by 28%. The mean muscle oxygen tension decreased by 19.2%. The number of hypoxic and anoxic zones in muscle tissue decreased significantly, in particular in red muscle. The maintenance of the fish in hypoxic waters for 15 days led to a rise of the content of cytochromes in muscle and a qualitative readjustment of the mitochondrial respiratory chain manifested in an increase of the content of cytochromes a and a3 and a decrease of cytochrome b. In discussion, literature data are presented which support the described regularity. Original Russian Text ? A. A. Soldatov, M. V. Savina, 2008, published in Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, 2008, Vol. 44, No. 5, pp. 508–512.     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

Antibody Immunodiversity: A Study on the Marked Specificity Difference Between Two Anti-Yeast Iso-1 Cytochrome c Monoclonal Antibodies Whose Epitopes Are Closely Related

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity. Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.     

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center

Isotope substitution of H₂O by ²H₂O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P⁺) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P⁺. In the case of H₂O medium these effects are observed at a lower hydration than in ²H₂O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H₂O, the rate of electron transfer from the nearest high-potential cytochrome heme to P⁺ is virtually independent of hydration within the P/P₀ range from 0.1 to 0.5. In samples hydrated in ²H₂O this rate is approximately 1.5 times lower than in H₂O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with ²H₂O is inhibited within this range of P/P₀, whereas in RC samples with H₂O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

Recombination reduction of photooxidized cytochrome c in reaction centers of Rhodopseudomonas viridis at low temperature

The temperature dependence of dark reduction of photooxidized cytochrome c was studied in isolated preparations of Rhodopseudomonas viridis reaction centers. Within the range from room temperature to 260 K this process was found to be mediated by thermal diffusion of exogenous donor molecules, whereas at lower temperatures photooxidized cytochrome is reduced as a result of indirect recombination with photoreduced primary quinone acceptor. Kinetic simulation allowed certain thermodynamic characteristics of this reaction to be calculated. To the first approximation, these characteristics correlate with the estimates obtained from the results of direct redox titration.