Role of Arg-166 in Yeast Cytochrome c1

Abstract

A systematic screen for dominant-negative mutations of the CYT1 gene, which encodes cytochrome c1, revealed seven mutants after testing ∼104 Saccharomyces cerevisiae strains 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 cytochrome cl, with diminished growth on glycerol medium and diminished respiration but with the normal or near normal level of cytochrome c1 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 c1 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 bc1 complex. Thus, the large size of the side chain at position 166 is critical for the function of cytochrome c1 but not for its assembly in the cytochrome bc1 complex.