Electron transfer in cyanobacterial photosystem I. I. Physiological and spectroscopic characterization of site-directed mutants in a putative electron transfer pathway from A₀ through A₁ to F_x

The Photosystem I (PSI) reaction center contains two branches of nearly symmetric cofactors bound to the PsaA and PsaB heterodimer. From the x-ray crystal structure it is known that Trp⁶⁹⁷_PsaA and Trp⁶⁷⁷_PsaB are π-stacked with the head group of the phylloquinones and are H-bonded to Ser⁶⁹²_PsaA and Ser⁶⁷²_PsaB, whereas Arg⁶⁹⁴_PsaA and Arg⁶⁷⁴_PsaB are involved in a H-bonded network of side groups that connects the binding environments of the phylloquinones and F_x. The mutants W697F_PsaA, W677F_PsaB, S692C_PsaA, S672C_PsaB, R694A_PsaA, and R674A_PsaB were constructed and characterized. All mutants grew photoautotrophically, yet all showed diminished growth rates compared with the wild-type, especially at higher light intensities. EPR and electron nuclear double resonance (ENDOR) studies at both room temperature and in frozen solution showed that the PsaB mutants were virtually identical to the wild-type, whereas significant effects were observed in the PsaA mutants. Spin polarized transient EPR spectra of the P₇₀₀⁺A₁^- radical pair show that none of the mutations causes a significant change in the orientation of the measured phylloquinone. Pulsed ENDOR spectra reveal that the W697F_PsaA mutation leads to about a 5% increase in the hyperfine coupling of the methyl group on the phylloquinone ring, whereas the S692C_PsaA mutation causes a similar decrease in this coupling. The changes in the methyl hyperfine coupling are also reflected in the transient EPR spectra of P₇₀₀⁺A₁^- and the CW EPR spectra of photoaccumulated A₁^-. We conclude that: (i) the transient EPR spectra at room temperature are predominantly from radical pairs in the PsaA branch of cofactors; (ii) at low temperature the electron cycle involving P₇₀₀ and A₁ similarly occurs along the PsaA branch of cofactors; and (iii) mutation of amino acids in close contact with the PsaA side quinone leads to changes in the spin density distribution of the reduced quinone observed by EPR.