PLANT PHYSIOLOGY , Vol 106, Issue 2 617-624, Copyright © 1994 by American Society of Plant Biologists
Identification of Surface-Exposed Domains on the Reducing Side of Photosystem I
Q. Xu, J. A. Guikema and P. R. Chitnis
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901
Photosystem I (PSI) is a multisubunit enzyme that catalyzes the
light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant
photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed
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 of 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 without significant
cleavage of other subunits. Glu63 and Glu67, located near the C terminus of
PsaE, were the most likely cleavage sites. At higher protease
concentrations, the PsaE fragments were further cleaved 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, several secondary cleavage sites on PsaD,
PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD
and PsaE were intact. Glu88 and Glu124 of PsaF became susceptible to
endoproteinase Glu-C upon extensive cleavage of PsaD and PsaE. Modification
of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase
Glu-C or thermolysin showed that the intact PsaE and PsaD, but not their
major degradation products lacking C-terminal domains, were heavily
biotinylated. Therefore, lysine-74 at the C terminus of PsaE was accessible
for biotinylation. Similarly, lysine-107, or lysine-118, or both in PsaD
could be modified by NHS-biotin.
