Beth Hacker, Blanca Barquera, A.R. Crofts and R.B. Gennis
Department of Biochemistry, and Program in Biophysics, University of Illinois at Urbana-Champaign
(This paper appeared in Biochemistry (1993) 32, 4403-4410)
In this work, site-directed mutagenesis has been used to explore residues in the cytochrome b subunit that might contribute to the quinone reducing site (Qc-site) of the bc1 complex of Rb. sphaeroides. The residues examined are Ala52 (Gly37), His217 (His202), Lys251 (Lys228) and Asp252 (Asp229) (numbers in parentheses are equivalent residues in yeast mitochondrial cytochrome b sequence). These residues were chosen either on the basis of sequence alignment with residues modified in antimycin resistance strains (G37V and K228I/M in yeast), or location of conserved residues close to the putative Qc-site (antimycin binding domain) (H202/217 and D229/252). The phenotypical consequences of these mutations were monitored by observing effects on spectra and midpoint potentials of the cytochromes, and on the kinetics of the electron transfer both to and from cytochrome bH following flash illumination. Since electrons transfer from cytochrome bH is thought to occur to either quinone of semiquinone bound at the quinone reducing site of the bc1 complex, analysis of the effects of the mutations should help map the location of residues which contribute to the quinol reducing site, and indicate the role of specific residues.
Reduction of cytochrome bH through quinol oxidation at the Qz-site was normal in all strains. Since at least one mutant modified at each of the other sites was able to grow photosynthetically, none of the residues was essential for the functioning of the quinol reductase site. However, each of the mutations resulted in effects indicating a severely or partially defective quinone reducing site.
Mutation of H217 to alanine resulted in a strain unable to grow photosynthetically, and with a severe block in cytochrome bH oxidation, suggesting an important role for this residue. All of the mutants have a reduced rate of the antimycin-sensitive carotenoid change as compared to BC17C, suggesting an impaired oxidation of cytochrome bH. Strains K251I and K251M show a change of similar magnitude to BC17C but with a reduced rate (K251I kinetics not shown). A52V and D252N have rates more markedly inhibited compared to BC17C, and an amplitude truncated compared to wild type. In mutants D252A and H217A, the electron transfer from cytochrome bH is so strongly inhibited that no antimycin sensitive carotenoid change was seen during the relatively short time span of the kinetic trace. The inhibition is most severe in strains D252A and H217A, which fail to grow photosynthetically, is quite marked in strain D252N and A52V, and relatively mild in strains K251M and K251I. All strains which show a significant rate of turn over of the bc1 complex, as judged by the antimycin sensitive carotenoid change, were able to grow photosynthetically.
The electrogenic reduction of cytochrome bH can be measured through the myxothiazol sensitive component of the change in the presence of antimycin. All strains showed a kinetics for this component of the electrogenic process similar to that of the BC17C strain. These results are in good agreement with the results from flash-induced kinetics of cytochrome bH redox changes, and show that the inhibition of electron flow in the mutant strains is localized at the step of cytochrome bH oxidation.
All mutants showed an antimycin induced oxidation of cytochrome b150, with a relatively tight binding of antimycin. Strains H217A and K251I showed end-points for the titration which were within experimental error the same as the wild type BC17C strain, and all three strains showed the sharp end-point characteristic of a tightly binding inhibitor. Strains K251M, A52V, D252N and D252A showed weaker binding, as indicated both by the higher titre, and by the curvature of the approach to the end-point of the titration. The most marked difference between strains was in the amplitude of the antimycin induced oxidation. This was most dramatic in H217A, where more than half the cytochrome bH titrated as b150, and became oxidized on addition of antimycin. However, all mutant strains apart from A52V showed an enhanced antimycin induced oxidation when compared to the wild type strain.