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Redox function and thermostability of various P. aeruginosa cytochrome c551 mutants
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Journal OfInorganic Biochemistry 96 (2003)
Redox function and thermostability of various P. aeruginosa cytochrome c,,~ mutants Yasuhiko Yamamoto, Department of Chemistry, University of Tsukuba, Japan Norifumi Terui, Department of Chemistry, University of Tsukuba, Japan Hajime Mita, Department of Chemistry, University df Tsukuba, Japan Yoshihiro Sambongi, Faculty ofApplied Biological Science, Hiroshima University, Japan Hikaru Hemmi, National Food Research Institute, Japan Susumu Uchiyama, Graduate School of Engineering, Osaka University, Japan Yuji Kobayashi, Graduate School of Pharmaceutical Science, Osaka University, Japan Yasuo Igarashi, Department of Biotechnology University of Tokyo, Japan Naoki Tachiiri, University of Tsukuba, Japan Shin-ich1 Joseph Takayama, University of Tsukuba, Japan Mesophile I? aeruginosa cytochrome c,,* (Pa) and thermophile H. thermophilus cytochrome c552(Ht) exhibit high sequence identity (56%) and their main-chain folding is almost identical. But Pa is considerably less stable than Ht. A series of Pa mutants, in which amino acid substitutions had been structurally selected with reference to the corresponding residues in Ht, exhibited increased stability compared with the wild-type Pa. Oxidized forms of the single mutant (F34Y) and quintuple mutant (F7AIV13M/F34Y/E43Y/V781) in the presence of 1.5 M guanidine hydrochloride exhibited the denaturation temperatures of 66.4”C and 80.2’C, respectively, as opposed to the value of 50.4”C for the wild-type Pa under the identical conditions. Electrochemical and NMR spectral properties for the proteins that differ in the overall protein stability have been characterized in detail. We found that the redox potential (E”‘) of the protein is correlated well with the protein stability and that the differences in the E”’ among the wild-type Pa and mutants are attributed predominantly to the enthalpic contribution to the E”‘. Temperature-dependent appearance of the NMR spectra of both oxidized and reduced forms of the proteins facilitated a direct comparison of the thermostability of the heme active site among the proteins. The higher stability of the reduced form than that of the oxidized one, for a given protein, was confirmed by the NMR study. Since only a small redox-dependent polypeptide conformation change has been reported for electron transfer proteins, the difference in the thermodynamic stability between the two different redox forms of the proteins should be attributed to structural properties of the heme active site, which are largely affected by the oxidation state of heme iron. The results strongly suggested that the stability of the heme coordination structure in the proteins regulates the thermodynamic stability of both the redox forms of the proteins, which in turn affects the E”‘.
Journal OfInorganic Biochemistry 96 (2003)
Redox function and thermostability of various P. aeruginosa cytochrome c,,~ mutants Yasuhiko Yamamoto, Department of Chemistry, University of Tsukuba, Japan Norifumi Terui, Department of Chemistry, University of Tsukuba, Japan Hajime Mita, Department of Chemistry, University df Tsukuba, Japan Yoshihiro Sambongi, Faculty ofApplied Biological Science, Hiroshima University, Japan Hikaru Hemmi, National Food Research Institute, Japan Susumu Uchiyama, Graduate School of Engineering, Osaka University, Japan Yuji Kobayashi, Graduate School of Pharmaceutical Science, Osaka University, Japan Yasuo Igarashi, Department of Biotechnology University of Tokyo, Japan Naoki Tachiiri, University of Tsukuba, Japan Shin-ich1 Joseph Takayama, University of Tsukuba, Japan Mesophile I? aeruginosa cytochrome c,,* (Pa) and thermophile H. thermophilus cytochrome c552(Ht) exhibit high sequence identity (56%) and their main-chain folding is almost identical. But Pa is considerably less stable than Ht. A series of Pa mutants, in which amino acid substitutions had been structurally selected with reference to the corresponding residues in Ht, exhibited increased stability compared with the wild-type Pa. Oxidized forms of the single mutant (F34Y) and quintuple mutant (F7AIV13M/F34Y/E43Y/V781) in the presence of 1.5 M guanidine hydrochloride exhibited the denaturation temperatures of 66.4”C and 80.2’C, respectively, as opposed to the value of 50.4”C for the wild-type Pa under the identical conditions. Electrochemical and NMR spectral properties for the proteins that differ in the overall protein stability have been characterized in detail. We found that the redox potential (E”‘) of the protein is correlated well with the protein stability and that the differences in the E”’ among the wild-type Pa and mutants are attributed predominantly to the enthalpic contribution to the E”‘. Temperature-dependent appearance of the NMR spectra of both oxidized and reduced forms of the proteins facilitated a direct comparison of the thermostability of the heme active site among the proteins. The higher stability of the reduced form than that of the oxidized one, for a given protein, was confirmed by the NMR study. Since only a small redox-dependent polypeptide conformation change has been reported for electron transfer proteins, the difference in the thermodynamic stability between the two different redox forms of the proteins should be attributed to structural properties of the heme active site, which are largely affected by the oxidation state of heme iron. The results strongly suggested that the stability of the heme coordination structure in the proteins regulates the thermodynamic stability of both the redox forms of the proteins, which in turn affects the E”‘.