Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c.
Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.
Schejter
A
A
Department of Biological Sciences, University of Illinois at Chicago 60637.
Koshy
T I
TI
Luntz
T L
TL
Sanishvili
R
R
Vig
I
I
Margoliash
E
E
D000818:Animals; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D003486:Cyanides / Q000378:metabolism; D003574:Cytochrome c Group / Q000235:genetics* / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007532:Isoleucine / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D003486:Cyanides; D003574:Cytochrome c Group; D007477:Ions; D007532:Isoleucine; D006418:Heme; D001216:Asparagine