Structure-function relationships of the mitochondrial bc1 complex in temperature-sensitive mutants of the cytochrome b gene, impaired in the catalytic center N.

Abstract

Seven new structures of cytochrome b have been recently identified by isolating and sequencing revertants from cytochrome b respiratory deficient mutants (Coppée, J. Y., Brasseur, G., Brivet-Chevillotte, P., and Colson, A. M. (1994) J. Biol. Chem. 269, 4221-4226). These mutations are located in the center N domain (QN). All the revertants exhibited a modified heme b562 maximum, confirming that part of the NH2-terminal region is in the vicinity of the extramembranous loop between helices IV-V and heme b562. Based on measurements performed on the maximal activities occurring in each segment of the respiratory chain, the decrease observed in the NADH oxidase activities of several revertants was correlated with some bc1 complex activity impairments; this may also explain why a moderate decrease in bc1 complex activity does not limit the succinate oxidase activity. The decrease in the rate of reduction of cytochrome b via the center N pathway is responsible for the impairment of the bc1 complex activity of these revertants. The three double-mutated revertants (S206L/N208K or -Y; S206L/W30C) are temperature-sensitive in vivo, and their mitochondria like that of the original mutant S206L are thermosensitive in vitro. Isolating the W30C mutation does not yield a thermosensitive phenotype: the replacement of serine 206 by leucine is therefore responsible for the thermoinstability of these strains; this temperature sensitivity is reinforced by additional mutations N208K or N208Y, and not by W30C. These data suggest that serine 206 and asparagine 208 are involved in the thermostability of the protein. When bc1 complex activity is lost after incubating mitochondria at a nonpermissive temperature (37 degrees C), heme b is still present, but can no longer be reduced by physiological substrate. The progressive loss of bc1 complex activity seems to be initially linked to a change in the tertiary structure of cytochrome b, which occurs drastically at center N and much more slowly at center P, as shown by kinetic study on the two cytochrome b redox pathways.