A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect.
A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the difference between the horse and yeast proteins was shown to be played by the amino acid residue in position 4 (glutamate in horse, and lysine in yeast; horse protein numbering). A mutant of the yeast cytochrome c containing K4E and some other "horse" modifications in the N-terminal alpha-helix, proved to be (i) much more active in electron transfer and antioxidant activity than the wild-type yeast cytochrome c and (ii), like the yeast cytochrome c, inactive in caspase stimulation, even if added in 400-fold excess compared with the horse protein. Thus this mutant seems to be a good candidate for knock-in studies of the role of cytochrome c-mediated apoptosis, in contrast with the horse K72R, K72G, K72L and K72A mutant cytochromes that at low concentrations were less active in apoptosis than the wild-type, but were quite active when the concentrations were increased by a factor of 2-12.
Abdullaev
Ziedulla Kh
ZKh
Laboratory of Spectral Analysis, M.M. Shemyakin and Y.A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow 177871, Russia.
Bodrova
Marina E
ME
Chernyak
Boris V
BV
Dolgikh
Dmitry A
DA
Kluck
Ruth M
RM
Pereverzev
Mikhail O
MO
Arseniev
Alexander S
AS
Efremov
Roman G
RG
Kirpichnikov
Mikhail P
MP
Mokhova
Elena N
EN
Newmeyer
Donald D
DD
Roder
Heinrich
H
Skulachev
Vladimir P
VP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D006736:Horses; D007700:Kinetics; D008930:Mitochondria, Liver / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D000975:Antioxidants; D003574:Cytochrome c Group; D011994:Recombinant Proteins; D020169:Caspases