International workshop on non-mitochondrial cytochromes c. A short foreword with some history.

A brief description of the informal workshop on non-mitochondrial cytochromes c is given. The organization of the meeting, the personnel participating and the scope of research effort are discussed. A brief historical account is included to place the Symposium reports in context.

Separation of respiratory reactions in Rhodospirillum rubrum: inhibition studies with 2-hydroxydiphenyl.

1. Respiration of chemotrophically and phototrophically grown Rhodospirillum rubrum is inhibited by 2-hydroxydiphenyl. 2. Membrane-bound NADH oxidase and NADH: cytochrome c reductase are inhibited also. The inhibitor constant for both reactions (Ki) is 0.075 plus or minus 0.012 mM. NADH dehydrogenase is not inhibited significantly. 3. The inhibition of succinate:cytochrome c reductase is associated for chemotrophic membranes with Ki equals 0.22 plus or minus 0.03 mM and for phototrophic membranes with Ki equals 0.49 plus or minus 0.09 mM. Succinate dehydrogenase is not affected by 2-hydroxydiphenyl. 4. Cytochrome oxidase is inhibited only slightly. 5. While NADH-dependent reactions in both phototrophic and chemotrophic membranes are inhibited maximally more than 95 percent, succinate-dependent reactions can be inhibited more than 95 percent only in chemotrophic membranes. In phototrophic membranes the maximum inhibition of succinate-dependent reactions is about 70 percent. 6. The type of inhibition in both cases 2 and 3 is non-competitive. 7. While the reduction of b-type cytochrome is inhibited by 2-hydroxydiphenyl, the degree of ubiquinone reduction is not influenced. The data suggest that the site of inhibition is localized between ubiquinone and cytochrome b. 8. Implications of these data for the respiratory electron transport system in R. rubrum are discussed.

Electron transport in an in vitro-reconstituted bacterial photophosphorylating system.

Photooxidation of endogenous cytochrome(s) c, photoreduction of endogenous chrome(s) b and photobleaching of bacteriochlorophyll have been demonstrated in an in vitro reconstituted system, previously demonstrated to support photophosphorylation. The kinetic responses of these redox reactions to substrate and antimycin A in these particle are characteristic of electron transport processes and stronglysupport the contention that all, or a part of, the oxidative phosphorylation electron transport pathway can be coupled to reaction center, photopigment complex in a manner which supports photophosphorylation. In addition, a succinate-supported light dependent reduction of NAD+ was found.

Chelating agents protect hydrogenase against oxygen inactivation.

The effect of chelation on rate or air inactivation of hydrogenase from Clostridium pasteurianum has been investigated. All chelating agents used, whether water-soluble or water-insoluble, afforded protection against oxygen inactivation. EDTA appeared to be the most effective. Thus, in the absence of EDTA, hydrogenase in aqueous solution was nearly totally inactivated after 1 hour incubation in air, whereas 0.5 M EDTA (which did not affect significantly catalytic activity) allowed 41% retention of the initial activity even after 3 days incubation.

Redox potentials of the photosynthetic bacterial cytochromes c2 and the structural bases for variability.

The cytochromes c2 of the Rhodospirillaceae show a much greater variation in redox potential and its pH dependence than the mitochondrial cytochromes c that have been studied. It is proposed that the range of redox potential for cytochromes c2 functioning as the immediate electron donor to photo-oxidised bacteriochlorophyll may be 345-395 mV at pH 5. Closely related cytochromes c2 with different redox potentials show patterns of amino acid substitution which are consistent with changes in hydrophobicity near the haem being at least a partial determinant of redox potential. More distantly related cytochromes are difficult to compare because of the large number of amino acid substitutions and the probability that there are subtle changes in overall peptide chain folding. The redox potential versus pH curves can be analysed in terms of either one ionisation in the oxidised form or two in the oxidised form and one in the reduced. The pK in the oxidised form at higher pH values can be correlated with the pK for the disappearance or shift of the near infrared absorption band located near 695 nm. The structural bases of these ionisations are not known but the possible involvement of the haem propionate residues is discussed.

The effect of electron carriers and other ligands on oxygen stability of clostridial hydrogenase.

The effects of various electron carriers, a substrate (H2) and a reversible inhibitor (CO) on the rate of irreversible oxygen inactivation of clostridial hydrogenase (ferredoxin: H+ oxidoreductase, EC 1.18.3.1) have been studied kinetically. Some electron carriers (e.g., clostridial ferredoxin and methyl viologen) greatly stabilize the enzyme, some (FAD, FMN) drastically reduce its stability, while others (benzyl viologen and methylene blue) only slightly alter the stability. Competitive experiments indicate that stabilizers and destabilizers do not compete with each other for binding with the active center of hydrogenase. Hydrogen and CO do not affect the rate of the oxygen inactivation. On the basis of the results obtained herein and kinetic data on hydrogenase catalysis from the literature, it is concluded that the active center of this hydrogenase comprises at least three different independent subsites. The first one (presumably an iron atom of the iron-sulfur cluster) binds H2 and CO and does not contribute to the oxygen stability. The second one binds stabilizers like methyl viologen while the third one binds destabilizers like FMN and FAD.

pH dependence of the oxidation-reduction potential of cytochrome c2.

The pH dependence of the spectra and of the oxidation-reduction potential of three cytochromes c2, from Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodomicrobium vannielii, were studied. A single alkaline pK was observed for the spectral changes in all three ferricytochromes. In Rps. capsulata cytochrome c2 this spectroscopic pK corresponds to the pK observed in the dependence of oxidation-reduction potential on pH. For the other two cytochromes the oxidation-reduction potential showed a complex dependency on pH which can be fitted to theoretical curves involving three ionizations. The third ionization corresponds to the ionization observed in the spectroscopic studies but the first two occur without changes in the visible spectra. The possible structural bases for these ionizations are discussed.

Amino acid sequences of cytochromes c2 and c' from the moderately halophilic purple phototrophic bacterium Rhodospirillum salexigens.

Rhodospirillum salexigens is a moderately halophilic purple phototrophic bacterium which grows optimally in 8% NaCl. The amino acid sequences of the two principal soluble cytochromes c have been determined. One of these is a cytochrome c2, similar in size to mitochondrial cytochrome c. While clearly of the same sequence class as mitochondrial cytochrome c and the proteins from several other Gram-negative bacteria, it does not show particular affinity to any already known sequence in terms of the percentage sequence identity. The other protein is a cytochrome c', but is also a divergent member of this widespread group. The lack of appreciable sequence identity to other species is probably due to a limit of divergence which has been reached for the majority of purple bacterial species. However, the numbers of insertions and deletions and their locations in cytochromes c2 and c' suggest that R salexigens may be related to Rhodospirillum molischianum. Like other electron transport proteins from halophiles, both of these cytochromes are notable for their high content of arginine as compared with lysine and both are acidic. However, they do not show any particular sequence homology to electron transport proteins that have been characterized from the extremely halophilic phototrophes of the genus Ectothiorhodospira. Thus, it appears that adaptation to halophilic habitats has independently occurred more than once in purple bacteria.