Role of Arg-166 in Yeast Cytochromec1*

A systematic screen for dominant-negative mutations of the CYT1gene, which encodes cytochromec1, revealed seven mutants after testing ∼104Saccharomyces cerevisiaestrains transformed with a library of mutagenized multicopy plasmids. DNA sequence analysis revealed multiple nucleotide substitutions with six of the seven altered Cyt1p having a common R166G replacement, either by itself or accompanied with other amino acid replacements. A single R166G replacement produced by site-directed mutagenesis demonstrated that this change produced a nearly nonfunctional cytochromec1, with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c1having an attached heme group. In contrast, R166K, R166M, or R166L replacements resulted in normal or near normal function. Arg-166 is conserved in all cytochromesc1and 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 cytochromebc1complex. Thus, the large size of the side chain at position 166 is critical for the function of cytochromec1but not for its assembly in the cytochromebc1complex.