PARENT SESSION

Symposium S6A Type II reaction centres: Excited state dynamics and donor side
Thursday September 2nd, 2004 10:20 AM-12:20 PM Room 511D
Chair: Bill Rutherford
Co-Chair: Marilyn Gunner

Changes in midpoint potentials of hemes by SD-mutagenesis in RC-bound tetraheme cytochrome of Blastochloris viridis. Kenji Nagashima^*,1, Jean Alric², Katsumi Matsuura¹, Keizo Shimada¹, Andre Vermeglio³, ¹ Department of Biology, Tokyo Metropolitan University, Minamiohsawa 1-1, Hachioji, Tokyo, Japan² Laboratoire de Génétique et Biophysique des Plantes, UMR 163 CEA-CNRS, Univ. Méditerranée-CEA 1000, 163 avenue de Luminy, Marseille, France³ CEA/Cadarache DEVM-Laboratoire de Bioénergétique Cellulaire, UMR 163-CNRS-CEA, Univ-Méditerranée CEA 1000, 13108 Saint Paul lez, France

ABSTRACT- A chimeric reaction center complex composed of L, M and H subunits of a purple bacterium, Rubrivivax gelatinosus, and the tetraheme cytochrome subunit of another purple bacterium, Blastochloris viridis, was successfully synthesized in R. gelatinosus cells, in which the original gene for the cytochrome subunit was deleted and replaced by that of B. viridis. The redox midpoint potentials (E_m's) and the redox difference spectra of four hemes in the cytochrome subunit of the chimeric RC were almost identical with those in the B. viridis RC, in which the hemes are linearly arranged from the LM core to the periplasmic space as c559 (+398 mV), c552 (+32 mV), c556 (+310 mV), and c553 (-60 mV). To clarify the physiological significance of such high-low-high-low arrangement of E_m's of hemes, acidic (Glu and Asp) and basic (Lys and Arg) amino acid residues located near the hemes of the B. viridis cytochrome subunit, which have been suspected to have important effects on tuning the redox midpoint potentials by Gunner and Honig (Proc. Natl. Acad. Sci. USA 1991, 88, 9151-9155) and Voigt and Knapp (J. Biol. Chem. 2003, 278, 51993-52001), were replaced by residues with the opposite charges, respectively. In addition, some neutral residues are replaced by charaged residues. More than 20 mutants were obtained by introduction of the B. viridis cytochrome gene mutations in the R. gelatinosus host cells to synthesize mutated chimeric reaction centers. In one of the mutants, R202E, in which the 202th Arg was replaced by Glu, c559 was redox-titrated as a low-potential heme with E_m of about 70 mV but the photooxidized P was rereduced within 10 s. A significant alteration in electrochemical characteristics of c559 was observed also in R264E mutation. These mutants grow photosynthetically. Despite significant changes in the E_m of c559, the electron transfer rate of the overall P⁺ reduction by the c556 seemed conserved, consistently with the ability of the bacterium to grow photosynthetically.

KEY WORDS: RC-bound tetraheme cytochrome c, electron transfer, purple bacteria, redox midpoint potential