Open AccessMutations of iso‐1‐cytochrome c at positions 13 and 90 Separate effects on physical and functional properties
Author(s) -
HUANG Yue,
BEESER Scott,
GUILLEMETTE J. Guy,
STORMS Reginald K.,
KORNBLATT Jack A.
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb18977.x
Subject(s) - cytochrome c , cytochrome , cytochrome c oxidase , cytochrome c1 , coenzyme q – cytochrome c reductase , cytochrome c peroxidase , cytochrome b , biochemistry , chemistry , cytochrome p450 reductase , cysteine , enzyme , gene , mitochondrion , mitochondrial dna
Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c . The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three‐dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso‐1‐cytochrome c ; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso‐1‐cytochrome c containing Arg13→Cys and Cys102→Thr mutations), [D90C, C102T]cytochrome c (iso‐1‐cytochrome c containing Asp90→Cys and Cys102→Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso‐1‐cytochrome c containing Arg13→Cys, Asp90→Cys, and Cys102→Thr mutations) are functional in vivo . Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c , whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c . The activity of cytochrome‐ c oxidase using cytochrome c was monitored polarographically. Compared to the wild‐type or [C102T]cytochrome c , which shows two kinetic phases with cytochrome‐ c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron‐transfer activity of the mutant cytochromes c is inhibited by Hg 2+ . The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c , even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90→Cys mutation while the activity changes follow those of the Arg13→Cys mutation.