Tryptophan-191----phenylalanine, a proximal-side mutation in yeast cytochrome c peroxidase that strongly affects the kinetics of ferrocytochrome c oxidation.
On the basis of X-ray structural information, it was previously proposed that tryptophan-191 of yeast cytochrome c peroxidase (CCP) may be important in determining the spectroscopic and catalytic properties of the enzyme [Edwards, S. L., Xuong, Ng. H., Hamlin, R. C., & Kraut, J. (1987) Biochemistry 26, 1503-1511]. By use of site-directed mutagenesis and an Escherichia coli expression system, a mutant phenylalanine-191 (F191) CCP was prepared in order to examine the effects of altering the H-bonding and pi-pi interactions that occur between Trp-191 and the iron-coordinated proximal His-175 in the parent enzyme. The F191 mutant enzyme exhibits a dramatic decrease (approximately 3000-fold at pH 7) in V0/e for catalysis of peroxide-dependent ferrocytochrome c oxidation, while V0/e for oxidation of ferrocyanide is decreased only 4.6-fold compared to that of the parent. The Fe3+/Fe2+ Em,7 and the stability of the oxyferryl center in the H2O2-oxidized mutant enzyme are relatively unaffected by the mutation, but the species responsible for a radical-like signal centered at g = 2.00 has been destabilized approximately 100-fold with respect to spontaneous decay. Steady-state kinetic assays as well as transient-state laser flash photolysis experiments utilizing flavin semiquinones as reductants indicate that the mutant CCP forms a complex with cytochrome c but the oxyferryl center in the oxidized enzyme is no longer able to be rapidly reduced by ferrocytochrome c. The most likely reasons for this kinetic behavior are either that new steric constraints exist in the mutant which impede relaxation of the iron center to the resting ferric state or that the indole ring of Trp-191 is important in a specific interprotein electron-transfer pathway that exists between the heme centers of CCP and cytochrome c.
Mauro
J M
JM
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Fishel
L A
LA
Hazzard
J T
JT
Meyer
T E
TE
Tollin
G
G
Cusanovich
M A
MA
Kraut
J
J
D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000235:genetics*; D010649:Phenylalanine; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry; D014364:Tryptophan
D003574:Cytochrome c Group; D010649:Phenylalanine; D014364:Tryptophan; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase