STRUCTURE-FUNCTION-RELATIONSHIPS OF THE MITOCHONDRIAL BC(1) COMPLEX IN TEMPERATURE-SENSITIVE MUTANTS OF THE CYTOCHROME-B GENE, IMPAIRED IN THE CATALYTIC CENTER-N

Seven new structures of cytochrome b have been recently identified by isolating and sequencing revertants from cytochrome b respiratory defi cient mutants (Coppee, J. Y., Brasseur, G., Brivet-Chevillotte, P., an d Colson, A.M. (1994) J. Biol, Chem. 269, 4221-4226). These mutations are located in the center N domain (Q(N)). All the revertants exhibite d a modified heme b(562) maximum, confirming that part of the NH2-term inal region is in the vicinity of the extramembranous loop between hel ices IV-V and heme b(562). Based on measurements performed on the maxi mal activities occurring in each segment of the respiratory chain, the decrease observed in the NADH oxidase activities of several revertant s was correlated with some b(c1) complex activity impairments; this ma y also explain why a moderate decrease in b(c1) 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 b(c1) complex activity of these revertants. The three double-mutated revertants (S206L/N208K or -Y; S206L/W30C) are te mperature-sensitive in vivo, and their mitochondria like that of the o riginal mutant S206L are thermosensitive in vitro. Isolating the W30C mutation does not yield a thermosensitive phenotype: the replacement o f serine 206 by leucine is therefore responsible for the thermoinstabi lity of these strains; this temperature sensitivity is reinforced by a dditional mutations N208K or N208Y, and not by W30C. These data sugges t that serine 206 and asparagine 208 are involved in the thermostabili ty of the protein. When b(c1) complex activity is lost after incubatin g mitochondria at a nonpermissive temperature (37 degrees C), heme b i s still present, but can no longer be reduced by physiological substra te. The progressive loss of b(c1) complex activity seems to be initial ly 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 s hown by kinetic study on the two cytochrome b redox pathways.