SITE-DIRECTED MUTAGENESIS OF CONSERVED HISTIDINES IN THE HELIX-VIII DOMAIN OF PSAB IMPAIRS ASSEMBLY OF THE PHOTOSYSTEM-I REACTION-CENTER WITHOUT ALTERING SPECTROSCOPIC CHARACTERISTICS OF P-700

The chloroplast psaB gene encodes one of the polypeptides of the photo system I reaction center heterodimer that coordinates the electron tra nsfer components P-700, A(0), and A(1). Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordina te the P-700 reaction center chlorophyll(s) and the initial electron a cceptor, A(0). Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosys tem I reaction center biogenesis and function. It is demonstrated that these histidine residues are essential for stable accumulation of the photosystem I reaction center. Protein pulse-labeling shows that chan ging the histidine residues impairs a post-translational step in react ion center assembly. Photosystem I complexes from the mutants have bee n characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P-700+. In all cases we determine that spectroscopic char acteristics of P-700+ remain unchanged. The implications of these resu lts to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.