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.

Dynamic Interaction of Plastocyanin with the Cytochrome bf Complex

“…At 25°C the value of the rate constant is 62 ϫ 10 6 M Ϫ1 s Ϫ1 in the presence of 150 mM NaCl. Assuming the same ionic strength dependence of the reaction rate at 5 (see below) and 25°C, this value extrapolates to a rate constant of 185 ϫ 10 6 M Ϫ1 s Ϫ1 at 90 mM NaCl, which is by a factor of 4 faster than that reported for truncated cyt f under these conditions (24,25,27) but comparable to the value of 100 ϫ 10 6 M Ϫ1 s Ϫ1 found recently (29). To analyze the surface regions of plastocyanin involved in the rate-limiting step of the reaction with the cyt bf complex, the role of residues in the hydrophobic and acidic patches of spinach plastocyanin was probed using site-directed mutagenesis.…”

“…Electrostatic Interaction-The effect of the ionic strength on the electron transfer from the cyt bf complex to plastocyanin indicates the attraction of two oppositely charged regions in the rate-determining step in good agreement with most studies using soluble cyt f and plastocyanin from heterologous (19,26) or homologous sources (29). It is not consistent with results showing no effect of mutated charges of residues 59 -61 (13,14) or an increase of the rate constant at increasing ionic strength below 40 mM (45) (cf.…”

“…A decrease of the rate constant determined by stopped-flow experiments after mutation of these residues to neutral or negative ones indicate their effect on the complex formation. However, in intact C. reinhardtii, these mutations showed only minor effects on the oxidation kinetics of cyt f after laser-induced oxidation of P700 (29). This difference is not fully understood but may partially be explained by the release of oxidized plastocyanin from PSI being rate-limiting for the oxidation of cyt f (18).…”

“…In the majority of these studies, the C-terminal trans-membrane helix of cyt f was removed to convert this cytochrome to a soluble protein. Furthermore, cyt f and plastocyanin were generally from heterologous sources, except for a recent study with the proteins from C. reinhardtii (29). Lee et al (13) have reported that exclusively mutations of the negative patch of residues 42-45 exerted an effect on the interaction with the solubilized cyt f. On the other hand, Kannt et al (26) found that both acidic patches are involved in this interaction and concluded that binding and not the rate of the intracomplex electron transfer is affected.…”

Electron Transfer from the Rieske Iron-Sulfur Protein (ISP) to Cytochrome f in Vitro

“…The lumenal and intramembrane domains of the cytochrome b 6 f complex of oxygenic photosynthesis contain four redox centers: cytochrome f with one covalently bound c-type heme, cytochrome b with two noncovalently bound b-type hemes, and one [2Fe-2S] cluster in the Rieske iron-sulfur protein (1)(2)(3). The electron transfer pathway, plastoquinol 3 Rieske ISP 1 3 cytochrome f 3 plastocyanin or cytochrome c 6 3 photosystem I on the lumen (p-side) of the membrane, comprises the high potential electron transport chain of the plastoquinol oxidase, whereas electron transfer between the two b-hemes, heme b p 3 heme b n , to a putative n-side bound quinone defines the low potential chain.…”