Amino acid substitutions at tryptophan-51 of cytochrome c peroxidase: effects on coordination, species preference for cytochrome c, and electron transfer

• Published in: Biochemistry (Easton) 1991-05, Vol.30 (20), p.4953-4962
• Main Authors: Goodin, David B, Davidson, Marilyn G, Roe, James A, Mauk, A. Grant, Smith, Michael
• Format: Article
• Language: English
• Subjects: activity; Amino Acid Sequence; amino acids; Analytical, structural and metabolic biochemistry; Base Sequence; Binding Sites; Biological and medical sciences; Cloning, Molecular; coordination; Cytochrome c Group - metabolism; cytochrome-c peroxidase; Cytochrome-c Peroxidase - genetics; Cytochrome-c Peroxidase - isolation & purification; Cytochrome-c Peroxidase - metabolism; Electron Spin Resonance Spectroscopy; electron transfer; Electron Transport; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; heme; Heme - metabolism; Kinetics; Mathematics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligonucleotide Probes; Oxidoreductases; Protein Conformation; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; replacement; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Spectrophotometry; substitution; Tryptophan
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00234a017

Amino acid replacements of an aromatic residue, Trp-51, which is in contact with the heme of yeast cytochrome c peroxidase have a number of significant effects on the kinetics and coordination state of the enzyme. Six mutants at this site (W51F, W51M, W51T, W51C, W51A, and W51G) were examined. Optical and EPR spectra show that each of these mutations introduces a shift from the 5-coordinate to 6-coordinate form, and slightly increases the asymmetry of the heme ligand field. Conversion from a 6-coordinate high-spin form at pH 5 to a 6-coordinate low-spin form at pH 7 is observed for several of the variants (W51F, W51T, and W51A), while W51G and W51C appear as predominantly low-spin species between pH 5 and 7. Addition of 50% glycerol prevents the facile conversion to the low-spin conformation for W51F, W51T, and W51A, and only W51F can be stabilized in a 5-coordinate configuration by glycerol. For the oxidation of cytochrome c by H2O2, three of the variants (W51F, W51M, and W51T) exhibit values of kcat(app) that are greater than for the wild-type enzyme, while the other mutations give decreased rates of enzyme turnover. Unlike the wild-type enzyme, which functions more efficiently with cytochrome c from yeast than with the horse heart protein, the mutant W51F does not show a preference for substrate from its native organism. The three mutants which exhibit increased values of kcat(app) show a pH optimum at 6.8 compared with that of 5.25 for the wild-type enzyme when measured with horse heart cytochrome c. This shift in pH optimum is not observed with yeast cytochrome c. Construction of single and multiple mutations at Trp-51, Ile-53, and Gly-152 shows that these kinetic properties are not due to natural amino acid variations observed at these sites. Pre-steady-state kinetics show that the bimolecular rate constant for the fast phase of the reaction of the enzyme with H2O2 is only slightly decreased from 3.03 (0.09) X 10(7) to 2.2 (0.1) X 10(7) M-1 s-1 for W51F and to 1.5 (0.1) X 10(7) M-1 s-1 for W51A. The slow phase of the reaction (4.9 s-1) which contributes approximately 30% to the amplitude of the change for the wild-type enzyme is not observed for W51F or W51A.