caa a22 4500 397494130 CHVBK 20180308164516.0 cr unu---uuuuu 161202e199506 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a124846 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a124846 Site-Directed Mutagenetic Study on the Role of Negative Patches on Silene Plastocyanin in the Interactions with Cytochrome f and Photosystem I [Elektronische Daten] [Byung Hyun Lee, Takashi Hibino, Tetsuko Takabe, Peter J. Weisbeek, Teruhiro Takabe] To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis. The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from −4 to +1. Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins. Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type. It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect. Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect. These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f. Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested. Based on these results, the electron transfer pathway from the heme to P700 via plastocyanin is discussed in relation to the role of charged amino acid residues. © Oxford University Press Regular Papers nationallicence copper protein nationallicence electron transfer nationallicence photosynthesis nationallicence plastocyanin nationallicence site-directed mutagenesis nationallicence Hyun Lee Byung BioScience Center, School of Agricultural Science, Nagoya University Chikusa-ku, Nagoya 464-01 aut Hibino Takashi Department of Chemistry, Faculty of Science & Technology, Meijo University Tenpaku-ku, Nagoya 468 aut Takabe Tetsuko BioScience Center, School of Agricultural Science, Nagoya University Chikusa-ku, Nagoya 464-01 aut Weisbeek Peter J. Institute of Molecular Biology and Department of Molecular Cell Biology, University of Utrecht Padualaan 8, 3584 CH Utrecht, The Netherlands aut Takabe Teruhiro Department of Chemistry, Faculty of Science & Technology, Meijo University Tenpaku-ku, Nagoya 468 aut The Journal of Biochemistry Oxford University Press 117/6(1995-06), 1209-1217 0021-924X 117:6<1209 1995 117 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a124846 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a124846 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hyun Lee Byung BioScience Center, School of Agricultural Science, Nagoya University Chikusa-ku, Nagoya 464-01 aut NATIONALLICENCE 700 1- Hibino Takashi Department of Chemistry, Faculty of Science & Technology, Meijo University Tenpaku-ku, Nagoya 468 aut NATIONALLICENCE 700 1- Takabe Tetsuko BioScience Center, School of Agricultural Science, Nagoya University Chikusa-ku, Nagoya 464-01 aut NATIONALLICENCE 700 1- Weisbeek Peter J. Institute of Molecular Biology and Department of Molecular Cell Biology, University of Utrecht Padualaan 8, 3584 CH Utrecht, The Netherlands aut NATIONALLICENCE 700 1- Takabe Teruhiro Department of Chemistry, Faculty of Science & Technology, Meijo University Tenpaku-ku, Nagoya 468 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 117/6(1995-06), 1209-1217 0021-924X 117:6<1209 1995 117 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford