Role of Arg-166 in yeast cytochrome c(1)

A systematic screen for dominant-negative mutations of the CYT1 gene, which encodes cytochrome c(1), revealed seven mutants after testing similar to 1 0(4) Saccharomyces cerevisiae strains transformed with a library of mutagen ized multicopy plasmids. DNA sequence analysis revealed multiple nucleotide substitutions with six of the seven altered Cyt1p having a common R166G re placement, either by itself or accompanied with other amino acid replacemen ts. A single R166G replacement produced by site-directed mutagenesis demons trated that this change produced a nearly nonfunctional cytochrome c(1), wi th diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c(1) having an attached heme group. In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function. Arg-166 is conserved in all cytochromes c(1) and lies on the surface of Cyt1p in close proximity to the heme group but does not seem to interact directly with any of the physiological partners of the cytochrome bc(1) complex. Thus, the large size of the side chain at positi on 166 is critical for the function of cytochrome c(1) but not for its asse mbly in the cytochrome bc(1) complex.