title: Identification of the basic residues of cytochrome f responsible for electrostatic docking interactions with plastocyanin in vitro: relevance to the electron transfer reaction in vivo.
abstract: The prominent basic patch seen in the atomic structure of the lumen-side domain of turnip cytochrome f, consisting of Arg209 and Lys187, 58, 65, and 66, was proposed to be an electrostatically complementary docking site for its physiological electron acceptor, plastocyanin [Martinez, S. E., Huang, D., Szczepaniak, A., Cramer, W. A., and Smith, J. L. (1994) Structure 2, 95-105]. This proposal agrees with solution studies on the cytochrome f/plastocyanin electron-transfer reaction that showed a major contribution of electrostatic interactions to the docking, but not with studies on the oxidation rate of cyt f in vivo using mutants in which the basic patch of cyt f was neutralized. The apparent contradiction might be explained by an unknown electron acceptor protein for cyt f. However, (i) flash-induced oxidation of cyt f is absent in a PC-deficient mutant. (ii) Lys58, 65, and 66 in the large domain and Lys188 and 189 in the small domain are major contributors to the ionic strength dependence of the electron-transfer reaction in solution. Replacement of Lys58 and 65 by neutral residues and of Lys66 by the acidic residue Glu66 resulted in a >10-fold decrease in the rate of electron transfer in solution and complete loss of its ionic strength dependence. Replacement of Lys188 and Lys189 in the small domain of cyt f resulted in a 3-4-fold decrease in the second-order rate constant and a smaller dependence of the overall rate of electron transfer on ionic strength, corresponding to a loss of two positive charges. (iii) Acidification of the thylakoid lumen cannot explain the absence of electrostatic interactions. (iv) Changing the five lysines to acidic residues did not result in any significant retardation of the rate of cyt f oxidation in vivo. If the docking of cyt f and plastocyanin in vivo is mediated by basic residues of cyt f, they are different from those that mediate electron transfer in vitro or that are implicated by simulations of electrostatic interactions of the docking. Alternatively, docking of cyt f/PC in vivo is limited by spatial constraints or release of PC from P700 that precludes a rate-limiting mediation of the cyt f/PC reaction by specific electrostatic interactions. The cyt f/PC system in Chlamydomonas reinhardtii is the first electron-transfer couple for which the role of electrostatics in mediating the docking reaction has been studied both in vitro and in vivo.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Soriano
authors.forename: M V
authors.initials: MV
authors.lastname: Ponamarev
authors.forename: R A
authors.initials: RA
authors.lastname: Piskorowski
authors.forename: W A
authors.initials: WA
authors.lastname: Cramer
mesh_terms: D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D002736:Chloroplasts / Q000201:enzymology; D003580:Cytochromes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D045348:Cytochromes f; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010970:Plastocyanin / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity
chemical_list: D003580:Cytochromes; D010970:Plastocyanin; D045348:Cytochromes f