Site-directed mutagenesis of yeast cytochrome c peroxidase shows histidine 181 is not required for oxidation of ferrocytochrome c.
The long-distance electron transfer observed in the complex formed between ferrocytochrome c and compound I, the peroxide-oxidized form of cytochrome c peroxidase (CCP), has been proposed to occur through the participation of His 181 of CCP and Phe 87 of yeast iso-1 cytochrome c [Poulos, T. L., & Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330]. We have examined the role of His 181 of CCP in this process through characterization of a mutant CCP in which His 181 has been replaced by glycine through site-directed mutagenesis. Data from single-crystal X-ray diffraction studies, as well as the visible spectra of the mutant CCP and its 2-equiv oxidation product, compound I, show that at pH 6.0 the protein is not dramatically altered by the His 181----Gly mutation. The rate of peroxide-dependent oxidation of ferrocytochrome c by the mutant CCP is reduced only 2-fold relative to that of the parental CCP, under steady-state conditions. Transient kinetic measurements of the intracomplex electron transfer rate from ferrous cytochrome c to compound I indicate that the rate of electron transfer within the transiently formed complex at high ionic strength (mu = 114 mM, pH = 6) is also reduced by approximately 2-fold in the mutant CCP protein. The relatively minor effect of the loss of the imidazole side chain at position 181 on the kinetics of electron transfer in the CCP-cytochrome c complex precludes an obligatory participation of His 181 in electron transfer from ferrous cytochrome c to compound I.(ABSTRACT TRUNCATED AT 250 WORDS)
Miller
M A
MA
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Hazzard
J T
JT
Mauro
J M
JM
Edwards
S L
SL
Simons
P C
PC
Tollin
G
G
Kraut
J
J
D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D005415:Flavins / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014961:X-Ray Diffraction
D003574:Cytochrome c Group; D005415:Flavins; D005656:Fungal Proteins; D006639:Histidine; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase