PARENT SESSION

Symposium S7A Mechanisms of water oxidation
Thursday September 2nd, 2004 2:40 PM-4:40 PM Room 210A
Chair: Stenbjörn Styring
Co-Chair: Ron Pace

Functional and structural properties of oxygen-evoling complex in D1 C-terminal mutants. Naoki Mizusawa^*,1, Yukihiro Kimura¹, Asako Ishii¹, Toshihiro Yamanari², Shigeaki Nakazawa¹, Haruhiko Teramoto¹, Taka-aki Ono¹, ¹ Laboratory for Photo-Biology (I), Photodynamics Research Center, RIKEN, 519-1399 Aoba, Sendai 980-0845, Japan² Faculty of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 739-8521, Japan

ABSTRACT- A free -COO^- of C-terminal Ala344 in the D1 protein has been proposed to be responsible for ligating the Mn cluster in oxygen-evolving complex (OEC). In order to address this issue, we constructed cyanobacterium Synechocystis sp. PCC 6803 mutants, in which Ala344 of the D1 protein was replaced by glycine, valine, aspartate, or aspargine (Ala344Gly-stop, Ala344Val-stop, Ala344Asp-stop, Ala344Asn-stop) in a background of a strain with a His-tag attached to CP47. Under moderate light conditions (50 Em^-2s^-1), all the mutants grew photoautotrophically, however, Ala344Gly-stop and Ala344Asp-stop mutants could not grow photoautotrophically at 200 Em^-2s^-1. Ala344Gly-stop and Ala344Asp-stop mutants exhibited thermoluminescence Q-band (S₂Q_A^-) and B-band (S₂Q_B^-) with elevated peak temperatures by 5^oC as compared with the control strain (Ala344-stop). Oxygen-evolving Photosystem II core particles purified from these mutants showed the same protein composition as that of the control core particles. The Gly, Asp, or Asn-substituted OEC showed smaller but normal multiline and enhanced g = 4.1 S₂ ESR signals, while Val-substituted OEC exhibited the largely normal ESR signals. The mid-frequency (1800 - 1000 cm^-1) S₂/S₁ Fourier transform infrared (FTIR) difference spectra showed relatively small but distinctive changes in the bands arising from the putative carboxylate ligands for the Mn cluster in all the mutants. The results indicate that the magnetic structure of the Mn-cluster and the structure of the carboxylate ligands are considerably altered by a change in the side group of the C-terminal amino acid residue of the D1 protein. Possible structural implication of the C-terminal D1-Ala344 in OEC is discussed.

KEY WORDS: site-directed mutagenesis, FTIR, Mn-cluster , ESR