title: Roles of charged residues in pH-dependent redox properties of cytochromec3fromDesulfovibrio vulgarisMiyazaki F.
abstract: Complicated pH-properties of the tetraheme cytochromec3(cytc3) fromDesulfovibrio vulgarisMiyazaki F (DvMF) were examined by the pH titrations of1H-15N HSQC spectra in the ferric and ferrous states. The redox-linked pKashift for the propionate group at C13 of heme 1 was observed as the changes of the NH signals around it. This pKashift is consistent with the redox-linked conformational alteration responsible for the cooperative reduction between hemes 1 and 2. On the other hand, large chemical shift changes caused by the protonation/deprotonation of Glu41 and/or Asp42, and His67 were redox-independent. Nevertheless, these charged residues affect the redox properties of the four hemes. Furthermore, one of interesting charged residues, Glu41, was studied by site-directed mutagenesis. E41K mutation increased the microscopic redox potentials of heme 1 by 46 and 34 mV, and heme 2 by 35 and 30 mV at the first and last reduction steps, respectively. Although global folding in the crystal structure of E41K cytc3is similar to that of wild type, local change was observed in1H NMR spectrum. Glu41 is important to keep the stable conformation in the region between hemes 1 and 2, controlling the redox properties ofDvMF cytc3. In contrast, the kinetic parameters for electron transfer fromDvMF [NiFe] hydrogenase were not influenced by E41K mutation. This suggests that the region between hemes 1 and 2 is not involved in the interaction with [NiFe] hydrogenase, and it supports the idea that heme 4 is the exclusive entrance gate to accept the electron in the initial reduction stage.
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka, Suita 565-0871, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Yahata
authors.affiliation: Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama 240-8501, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Ozawa
authors.affiliation: Graduate School of Life Science, University of Hyogo and Himeji Institute of Technology, Koto, Kamigori, Hyogo 678-1297, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tomimoto
authors.affiliation: Graduate School of Life Science, University of Hyogo and Himeji Institute of Technology, Koto, Kamigori, Hyogo 678-1297, Japan.
authors.forename: Kumiko
authors.initials: K
authors.lastname: Morita
authors.affiliation: Graduate School of Life Science, University of Hyogo and Himeji Institute of Technology, Koto, Kamigori, Hyogo 678-1297, Japan; RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Komori
authors.affiliation: Graduate School of Life Science, University of Hyogo and Himeji Institute of Technology, Koto, Kamigori, Hyogo 678-1297, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Ogata
authors.affiliation: Graduate School of Life Science, University of Hyogo and Himeji Institute of Technology, Koto, Kamigori, Hyogo 678-1297, Japan; RIKEN SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka, Suita 565-0871, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Akutsu
keywords: electron transfer; hydrogenase; nuclear magnetic resonance (NMR); site-directed mutagenesis; tetraheme protein