Abstract
Cellular localization of cytochrome (cyt) c550, a low potential, c-type monoheme cytochrome, in a thermophilic cyanobacterium Synechococcus vulcanus was investigated by systematic fractionation of the cells followed by its enzymatic and immunological detection. While cyt c-553, a soluble cyt that donates an electron to P700, was detected in the supernatant of osmotic disruption of lysozyme-treated cells, cyt c550 was detected only in the thylakoid membrane fraction, being tightly bound to thylakoids, and its removal required sonication in the presence of 1 M CaCl2. Upon further fractionation of the thylakoids into photosystem (PS) I and PSII by lauryl dimethylamine N-oxide solubilization, cyt c550 was exclusively concentrated in the crude PSII fraction together with cyt f, with no significant amount being detected in any of the soluble and PSI fractions. Upon further fractionation of the crude PSII by n-dodecyl beta-D-maltoside solubilization followed by column chromatography, cyt c550 was detected exclusively in the purified PSII core complex fraction but not in any other fractions. A 12-kDa protein, one of the extrinsic components of cyanobacterial PSII, exhibited completely the same behavior as that of cyt c550 during these fractionation procedures. These results, coupled with our previous results that cyt c550 binds stoichiometrically to the cyanobacterial PSII core complex and enhances O2 evolution (Shen, J.-R., and Inoue, Y. (1993) Biochemistry 32, 1825-1832), indicate that there is only one species of cyt c550 in cyanobacterial cells and that this cyt is exclusively associated with PSII as a functional component for O2 evolution.
Highlights
From the Solar Energy Research Group, The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan
They observed that isolated cyt c650 could be tightly bound to thylakoids, and its removal required reduced in vitro by ferredoxin I1 and proposed that cyt c550 sonication in the presenceof 1M C a C L Upon further may function in removing excess electrons generated by anfractionation of the thylakoids intpohotosystem (PS)I aerobic fermentation,thereby enabling the cells to survive and PSII by lauryl dimethylamine N-oxide solubiliza- under anaerobic conditions (5, 6)
Uponfurther fractionationof the crudePSII by n-dodecyl @-D-maltosidesolubilization followed by column chromatography, cyt csso was detected exclusively in the purified PSII core complex fraction but of Anacystis nidulam and proposed that cyt c550 may be an endogenous cofactor for cyclic electron transfer around PSI
Summary
The resulting supernatant was centrifuged a t 200,000 X g for 17 h to collect crude PSII. While this procedure gave rise to a rather pure PSI fraction withlittlePSIIcontamination,theresultingPSIIstillcontained. The crude PSII con- (panel A , lanes 1 and 2) represent soluble cytoplasmic comtained some other co-purifying components and a large amount of ponents, whereas those detected in the membrane fraction. Each fraction was numbered consecutively and subjectedband below PB proteins in lane I is lysozyme employed to to analysis for cyt cm (see "Results"). It is of note that this staining method selectively probes ctype cyts on ourgels, since b-type cytswould lose their heme during electrophoresis due to the presence of a high concentration of urea(14). -. - distinct bandin the 9-kDaregion in the supernatant obtained afterosmoticshocktreatment(panel R , l a w 2 ) andtwo bands in the 28- and 17-kDaregions in the thylakoid fraction
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