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Research has been conducted on antimycin-treated chromophores from Rodobacter sphaeroides. Results demonstrate that addition of myxothiazol to these chromophores at ambient redox potentials increases flash-induced oxidation of cytochrome c (sub)1.

Research examines the antimycin A- and/or myxothiazol-resistant reduction of cytochrome c with respect to side reactions, during Q-cycle catalysis, involving reduction of oxygen by the Q(sub)o site semiquinone, in isolated yeast cytochrome bc(sub)1 complex under both aerobic and anaerobic conditions.

Results show that the electrogenic reactions of the cytochrome bc(sup)1 complex are influenced by the transmembrane electric field produced by the reaction center of Rhodobacter sphaeroides. Data indicate that a reversal of electron transfer between b(sub)1 and b(sub)H hemes is a result of redox reactions associated with the b(sub)H heme.

Results show that Rhodobacter sphaeroides chromatophores are modified by N,N'-dicyclohexylcarbodiimide with changes in the rate of Q(sub)0H(sub)2 oxidation by the iron-sulfur protein.

Examination of the electrogenic and electron-transfer activities of the cytochrome bc1 complex in chromatophores from wild and mutant Rhodobacter sphaeroides bacteria, in which the cytochrome b aspartate-187 substitutes for asparagine, no difference in the inhibition patern by N,N'-dicyclohexylcarbodiimide is observed suggesting that aspartate-187 has no role in the protonogenic reaction.

Specific mutagenesis of the Rieske iron-sulfur protein in Rhodobacter sphaeroides is discussed. It shows that the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(sub.1) complex. Four mutant strains of Rb. sphaeroides were generated. In these strains Tyr 156 was mutated to Leu, His, Trp or Phe. The bc(sub.1) complex was put together and was able to support photosynthetic growth in mutants.

The movement of the iron sulfur subunit of mitochondrial ubihydroquinone:cytochrome c oxidoreductase is not responsible for the activation barrier associated with quinol oxidation. The activation energies of the reactions causing the movement are lower than the barrier.

The Q(sub)o site of mitochondrial ubihydroquinone:cytochrome C oxidoreductase can contain only an inhibitor or a quinol but not both. The exit tunnel forces the hydrophobic tails of compounds to occupy the same space. Therefore one would be displaced by the other.

The [2Fe-2S] cluster in ubihydroquinone:cytochrome c oxidoreductase rotates so the same docking surface interacts with cytochrome c(sub)1 and cytochrome b. The rotation occurs via an extension of a helical segment in the short linking span.

Quinol oxidation by the [bc.sub.1] complex of Rhodobacter sphaeroides occurs from an enzyme-substrate complex formed between quinol bound at the [Q.sub.o] site and the iron-sulfur protein (ISP) docked at an interface on cytochrome b. From the structure of the stigmatellin-containing mitochondrial complex, we suggest that hydrogen bonds to the two quinol hydroxyl groups, from Glu-272 of cytochrome b and His-161 of the ISP, help to stabilize the enzyme-substrate complex and aid proton release. Reduction of the oxidized ISP involves H transfer from quinol. Release of the proton occurs when the acceptor chain reoxidizes the reduced ISP, after domain movement to an interface on cytochrome [c.sub.1]. Effects of mutations to the ISP that change the redox potential and/or the pK on the oxidized form support this mechanism. Structures for the complex in the presence of inhibitors show two different orientations of Glu-272. In stigmatellin-containing crystals, the side chain points into the site, to hydrogen bond with a ring hydroxyl, while His-161 hydrogen bonds to the carbonyl group. In the native structure, or crystals containing myxothiazol or [Beta]-methoxyacrylate-type inhibitors, the Glu-272 side chain is rotated to point out of the site, to the surface of an external aqueous channel. Effects of mutation at this residue suggest that this group is involved in ligation of stigmatellin and quinol, but not quinone, and that the carboxylate function is essential for rapid turnover. [H.sup.+] transfer from semiquinone to the carboxylate side chain and rotation to the position found in the myxothiazol structure provide a pathway for release of the second proton.

A study was conducted to characterize two novel properties of cytochrome b6f complexes by altering the cytochrome b subunit of the cytochrome bc1 complex from Rhodobacter spaeroides. Site-directed mutagenesis was carried out using polymerase chain reaction techniques. Results indicated that threonine had little effect on protein folding or assembly. Findings also showed that the complementation between two fragments of a split cytochrome b supported a final conformation that accommodated rapid electron transfers through the mutant bc1 complex.

It has been established that the extrinsic domain of the Rieske protein containing the iron-sulphur cluster in cytochrome bc1, one of the three main respiratory enzyme complexes present in the inner mitochondrial membrane, assumes one of two conformations in the complexes. In one conformation, the iron-sulphur cluster is close to its electron acceptor, the haem group of cytochrome c1, but distant from the assumed binding site of its electron donor, ubiquinol, in cytochrome b. In the other, the cluster is closer to cytochrome b and further away from cytochrome c1. It was possible to identify the binding sites for two Qo-site inhibitors, stigmatellin and myxothiazol.

The role of isocytochrome type c2 (isocyt c2) in photosynthetic electron transfer chain involved in the transduction of light into biological energy was studied using kinetic assay. The isocyt c2 exhibited significantly lower affinity for RC complexes than is expected of the members of the cyt c2 family. It also appeared to limit the rate of P280+ reduction. These activities can be attributed to the sequence, structural and conformational properties of the cytochrome.

Site-directed mutagenesis of the highly conserved tryptophan-129 and arginine-14 residues in cytochrome b subunit of Rhodobacter sphaeroides bc1 complex reveals that the two residues are vital for the stability and structure of the bc1 complex. Mutations affect the quinone reductase site, cytochromes bH and bL and the photosynthetic development of R. sphaeroides. The functions of the two residues are in agreement with the positions of the residues close to the cytoplasmic sides of the putative transmembrane helices C and B.