Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin
Cytochrome P450cam (CYP101) of Pseudomonas putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐depe...
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Published in | FEBS letters Vol. 331; no. 1-2; pp. 109 - 113 |
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Main Authors | , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
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Amsterdam
Elsevier
27.09.1993
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Abstract | Cytochrome P450cam (CYP101) of Pseudomonas
putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid‐point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen‐bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd. |
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AbstractList | Cytochrome P450cam (CYP101) of Pseudomonas
putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate‐dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid‐point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen‐bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd. Cytochrome P450cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd. |
Author | Ishimura, Yuzuru Makino, Ryu Horiuchi, Tadao Shimada, Hideo Tsujimura, Mitsushi Yaoi, Tsuyoshi Yura, Kei Sekimizu, Kazuhisa Ikeguchi, Masamichi Koga, Hideo Nakamura, Kazuhide Sagara, Yasuhiro Go, Mitiko |
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Keywords | Pseudomonadales Hemoprotein Cytochrome P450 Electron transfer Bacteria Pseudomonadaceae Pseudomonas putida Structure function relationship |
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Snippet | Cytochrome P450cam (CYP101) of Pseudomonas
putida PpGl in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA... Cytochrome P450cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA... |
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SubjectTerms | Amino acid substitution Arg112, arginine residue at the position 112 of P450cam Arg112Cys, a mutant in which Arg112 has been changed by Cys Arginine - chemistry Bacterial Proteins - chemistry Bacteriology Binding of P450cam with putidaredoxin Biological and medical sciences Camphor 5-Monooxygenase Cytochrome P-450 Enzyme System - chemistry Cytochrome P-450 Enzyme System - genetics Cytochrome P450cam Electron transfer Electron Transport Ferredoxins - chemistry Fundamental and applied biological sciences. Psychology Metabolism. Enzymes Microbiology Mixed Function Oxygenases - chemistry Mixed Function Oxygenases - genetics Mutagenesis Oxidation-Reduction P450cam, cytochrome P450cam Pd, putidaredoxin PdR, putidaredoxin reductase Protein Conformation Pseudomonas putida - enzymology Putidaredoxin Random mutagenesis SDS-PAGE, sodium dodecyl sulfate/polyacrylamide gel electrophoresis |
Title | Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin |
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