IDENTIFICATION OF SURFACE-EXPOSED DOMAINS ON THE REDUCING SIDE OF PHOTOSYSTEM-I

Photosystem I (PSI) is a multisubunit enzyme that catalyzes the light- driven oxidation of plastocyanin or cytochrome cb and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-ex posed domains in PSI of the cyanobacterium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure o f PSI complexes to a low concentration of endoproteinase glutamic acid (Glu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments wit hout significant cleavage of other subunits. Glu(63) and Glu(67), loca ted near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cle aved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Glu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, sever al secondary cleavage sites on PsaD, PsaE, and PsaF were also identifi ed. PsaF resisted proteolysis when PsaD and PsaE were intact. Glu(88) and Glu(124) of PsaF became susceptible to endoproteinase Glu-C upon e xtensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase Glu-C or thermoly sin showed that the intact PsaE and PsaD, but not their major degradat ion products lacking C-terminal domains, were heavily biotinylated. Th erefore, lysine-74 at the C terminus of PsaE was accessible for biotin ylation. Similarly, lysine-107, or lysine-118, or both in PsaD could b e modified by NHS-biotin.