Site-directed photochemical coupling of cytochrome b6f-associated chlorophyll.
Cytochrome b(6)f complexes contain a molecule of chlorophyll a (Chla), which, in Chlamydomonas reinhardtii, can be exchanged for extraneous chlorophyll during protracted incubation of the purified complex in detergent solution. The specificity of the site and its location in the complex have been studied by photochemical coupling and circular dichroism spectroscopy. Following substitution of the original chlorophyll with [(3)H]Chla, the complex was irradiated in the Soret absorption band of Chla to complete bleaching and the amount of radioactivity covalently bound to each b(6)f subunit determined. Strong labeling was found to be associated with cytochrome f. The labeling originates from [(3)H]Chla molecules bound to a slowly exchanging site and showing the properties of the endogenous Chl, not from molecules dissolved in the detergent belt surrounding the complex. Chlorophyll b (Chlb) can compete with Chla, albeit with a lower affinity. Irradiation of [(3)H]Chlb introduced into the slowly exchanging site yielded the same labeling pattern that was observed with [(3)H]Chla. Proteolytic cleavage showed [(3)H]Chla labeling to be strictly restricted to the C-terminal region of cytochrome f. Circular dichroism spectra of the native complex revealed a bilobed signal characteristic of excitonic interaction between chlorophylls. The structural and evolutionary implications of these findings are discussed.
Pierre
Yves
Y
Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099, CNRS, and Université Paris-7, Institut de Biologie Physico-Chimique, CNRS IFR 550, 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Chabaud
Elodie
E
Hervé
Paulette
P
Zito
Francesca
F
Popot
Jean-Luc
JL
D000818:Animals; D002499:Centrifugation, Density Gradient; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000378:metabolism; D002734:Chlorophyll / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000077194:Chlorophyll A; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D045346:Cytochrome b6f Complex; D019281:Dimerization; D006868:Hydrolysis; D008027:Light; D010206:Papain / Q000378:metabolism; D010777:Photochemistry / Q000379:methods; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D020524:Thylakoids / Q000201:enzymology / Q000378:metabolism; D014316:Tritium
D003573:Cytochrome b Group; D021122:Protein Subunits; D014316:Tritium; D002734:Chlorophyll; C037184:chlorophyll b; D045346:Cytochrome b6f Complex; D010206:Papain; D000077194:Chlorophyll A