Hans Boumans

DIFFERENTIAL INHIBITION OF THE YEAST BC1 COMPLEX BY PHENANTHROLINES AND FERROIN : IMPLICATIONS FOR STRUCTURE AND CATALYTIC MECHANISM

o-Phenanthroline andm-phenanthroline both inhibit the electron transfer activity of lauryl maltoside-solubilized yeastbc 1 complex progressively with time. Pre-steady-state kinetics indicate that these compounds bind to the complex on the intermembrane space side, thereby blocking reduction of cytochrome b via the ubiquinol oxidation site.o-Phenanthroline is additionally capable of chelating an iron atom derived from the Rieske Fe-S cluster, thereby distorting the structure of the Rieske protein. EPR analysis shows that the secondary effect of o-phenanthroline occurs after initial inactivation and that m-phenanthroline, which lacks chelating activity, does not affect the Rieske Fe-S cluster. Spectral analysis shows that the b and c 1cytochromes are still dithionite-reducible after inactivation byo-phenanthroline, indicating that they remain intact. Inactivation by o-phenanthroline can be prevented by the addition of Fe2+. Surprisingly, ferroin, theo-phenanthroline-ferrous sulfate complex, also inhibits thebc 1 complex activity. In contrast too-phenanthroline, this effect is instantaneous. The two types of inhibition are clearly distinguishable by pre-steady-state reduction kinetics. Interestingly, ferroin can only inhibit electron transfer activity by about 50%. This behavior is discussed in relation to the dimeric structure of the bc 1 complex, and we conclude that ferroin binds to only one of the two protomers. The rate of inactivation by o-phenanthroline is dependent on the incubation temperature and can be quantitated in terms of the half-life for a certain temperature, the time at which thebc 1 activity is reduced to 50%. In contrast to the solubilized form, the bc 1 complex in intact mitochondria is insensitive to o-phenanthroline, suggesting that the inactivation rate by o-phenanthroline is dependent on accessibility of the complex to the agent. Reaction witho-phenanthroline is thus a useful technique for study of structural stability of the bc 1 complex under different conditions and should provide a sensitive tool for determination of the relative stability of mutant enzymes.

cDNA sequence of subunit VIII of ubiquinol-cytochrome-c oxidoreductase from Schizosaccharomyces pombe

We have cloned a cDNA coding for subunit VIII of the ubiquinol-cytochrome-c oxidoreductase of Schizosaccharomyces pombe by functional complementation of the null mutant in the QCR8 gene of Saccharomyces cerevisiae. DNA sequence analysis reveals an open-reading frame of 276 bp encoding a 10.5 kDa protein with 51% amino acid sequence identity to its counterpart in S. cerevisiae.

Identification of additional homologues of subunits VII and VIII of the ubiquinol-cytochrome c oxidoreductase enables definition of consensus sequences

The Candida utilis QCR7 gene encoding subunit VII of the ubiquinol‐cytochrome c oxidoreductase was isolated by functional complementation of the Saccharomyces cerevisiae subunit VII‐null mutant. Several other subunit VII homologues as well as homologues for subunit VIII were identified by screening the GenBank database. Some of these homologues for subunit VII could only be identified as such using a consensus sequence that was derived from the multiple sequence alignment. Definition of the consensus should facilitate further analysis of structure/function relationships in this protein.

Topological organization of subunits VII and VIII in the ubiquinolcytochrome c oxidoreductase of Saccharomyces cerevisiae

To determine the topology of subunit VIII of the yeast ubiquinol‐cytochrome c oxidoreductase in the mitochondrial inner membrane, an epitope has been introduced in the N‐terminal half of this protein. Previous topology studies had shown that at least the C‐terminus faces the intermembrane space [Hemrika and Berden (1990) Eur. J. Biochem. 192, 761–765]. Based on sensitivity of the protein to proteinase K digestion we now suggest that the N‐terminus of subunit VIII is similarly oriented, implying that this subunit does not span the membrane. Despite this, however, subunit VIII cannot be extracted from the membrane even after treatment with 0.1 M Na2CO3 at pH 11.5, showing that the protein is integrally embedded in the membrane. A similar behaviour was displayed by another low molecular weight protein of the complex, subunit VII, which faces the matrix side. A model for the topology of these subunits in the membrane is discussed with respect to the structure of the complex and their involvement in quinone binding.