Influence of the axial ligands on the spectral properties of P700 of photosystem I: A study of site-directed mutants

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) m olecules which form the primary electron donor (P700) of photosystem I (PSI ). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas r einhardtii by site-directed mutagenesis with nonpolar, uncharged polar, aci dic, and basic amino acid residues. Only the substitutions with uncharged p olar residues led to a significant accumulation of PSI in the thylakoid mem branes. These PSI complexes were isolated and the physical properties of th e primary donor characterized. The midpoint potential of P700(+.)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the ligh t-minus-dark absorbance spectra, all mutations in PsaB exhibited an additio nal bleaching band at 665 nm at room temperature comparable with the publis hed spectrum for the replacement of His(B656) with asparagine [Webber, A. N ., Su Hui, Bingham, S. E., Kass, H., Krabben, L., Kuhn, M., Jordan, R., Sch lodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substituti ons of His(A676) showed an additional shoulder around 680 nm. In the low-te mperature absorbance difference spectra of P700(+.)/P700, a blue shift of t he main bleaching band by 2 nm and some changes in the spectral features ar ound 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Simil ar effects of the mutations were found in the (3)P700/P700 absorbance diffe rence spectra at low temperatures (T = 2 K). The zero-field splitting param eters of (3)P700 were not significantly changed in the mutated PSI complexe s. The electron spin density distribution of P700(+.), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurem ents, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the muta tion of His(A676). (ii) The exchange of His(B656) with glutamine induces th e smallest changes in the spectra or the midpoint potential, whereas the ot her replacements exhibited a stronger but very similar influence on the spe ctroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are m ainly localized on the Chl a of the dimer which is axially coordinated by H is(B656).