Mutational analysis of photosystem I polypeptides: Role of PsaD and the LYSYL 106 residue in the reductase activity of photosystem I

The ADC4 mutant of the cyanobacterium Synechocystis sp. PCC 6803 was studied to determine the structural and functional consequences of the absence of PsaD in photosystem I. Isolated ADC4 membranes were shown to be deficient in ferredoxin-mediated NADP⁺ reduction, even though charge separation between P700 and F_A/F_B occurred with high efficiency. Unlike the wild type, F_B became preferentially photoreduced when ADC4 membranes were illuminated at 15 K, and the EPR line shapes were relatively broad. Membrane fragments oriented in two dimensions on thin mylar films showed that the g tensor axes of F_A^- and F_B^- were identical in the ADC4 and wild type strains, implying that PsaC is oriented similarly on the reaction center. PsaC and the F_A/F_B iron-sulfur clusters are lost more readily from the ADC4 membranes after treatment with Triton X-100 or chaotropic agents, implying a stabilizing role for PsaD. The specific role of Lys¹⁰⁶ of PsaD, which can be crosslinked to Glu⁹³ of ferredoxin (Lelong et al. (1994) J. Biol. Chem. 269, 10034-10039), was probed by site-directed mutagenesis. Chemical cross-linking and protease treatment experiments did not reveal any drastic alterations in the conformation of the mutant PsaD proteins. The EPR spectra of F_A and F_B in membranes of the Lys¹⁰⁶ mutants were similar to those of the wild type. Membranes of all Lys¹⁰⁶ mutants showed wild type rates of flavodoxin reduction and flavodoxin-mediated NADP⁺ reduction, but had 10-54% decrease in the ferredoxinmediated NADP⁺ reduction rates. This implies that Lys¹⁰⁶ is a dispensable component of the docking site on the reducing side of photosystem I and an ionic interaction between Lys¹⁰⁶ of PsaD and Glu⁹³ of ferredoxin is not essential for electron transfer to ferredoxin. These results demonstrate that PsaD serves distinct roles in modulating the EPR spectral characteristics of F_A and F_B, in stabilizing PsaC on the reaction center, and in facilitating ferredoxin-mediated NADP⁺ photoreduction on the reducing side of photosystem I.