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 P700

The chloroplast psaB gene encodes one of the polypeptides of the photosystem I reaction center heterodimer that coordinates the electron transfer components P₇₀₀, A₀, and A₁. Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordinate the P7oq reaction center chlorophyll(s) and the initial electron acceptor, A₀. Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosystem 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 changing the histidine residues impairs a post-translational step in reaction center assembly. Photosystem I complexes from the mutants have been characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P₇₀₀⁺. In all cases we determine that spectroscopic characteristics of P₇₀₀⁺ remain unchanged. The implications of these results to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.