MUTATIONAL ANALYSIS OF PHOTOSYSTEM-I POLYPEPTIDES IN SYNECHOCYSTIS SPPCC-6803 - SUBUNIT REQUIREMENTS FOR REDUCTION OF NADP(+) MEDIATED BY FERREDOXIN AND FLAVODOXIN

The subunit requirements NADP(+) reduction by photosystem I were asses sed in mutants of Synechocystis sp PCC 6803 created by targeted inacti vation of the psaD, psaE, psaF, and psaL genes. The PsaE-less, PsaF-Ps aJ-less, and PsaL-less mutants showed normal photoautotrophic growth, while the growth of PsaD-less mutants was slower without glucose. In i solated wild-type membranes, the rate of flavodoxin reduction and flav odoxin-mediated NADP(+) reduction were 800 and 480 mu mol/mg of chloro phyll/h, respectively. The rate of ferredoxin-mediated NADP(+) photore duction was 460 mu mol/mg of chlorophyll/h. There was no diminution in NADP(+) photoreduction in membranes isolated from the PsaF-less and P saL-less mutants. The rates of ferredoxin-mediated NADP(+) photoreduct ion in membranes of the PsaE-less mutants were 25 mu mol/mg of chlorop hyll/h. However, the rate of flavodoxin reduction was 380 mu mol/mg of chlorophyll/h, and that of flavodoxin-mediated NADP(+) photoreduction was 170 mu mol/mg of chlorophyll/h. PsaD-less membranes showed <20% o f the wild-type rates of flavodoxin-mediated NADP(+) photoreduction, b ut were completely deficient in ferredoxin-mediated NADP(+) photoreduc tion. Therefore, the roles of PsaE and PsaD are more crucial for ''doc king'' of ferredoxin than of flavodoxin. Proteolysis studies showed th at while PsaD was susceptible to rapid in vitro degradation by thermol ysin, the number and sizes of protease-resistant fragments were not af fected by the absence of PsaE. Protease accessibility studies further indicated that the C-terminal domain of PsaD is surface-exposed on the n-side. These results suggest that PsaE and the C-terminal domain of PsaD generate the docking site for the electron acceptors of photosyst em I.