EPR investigation of Cu2+-substituted photosynthetic bacterial reaction centers: Evidence for histidine ligation at the surface metal site

The coordination environments of two distinct metal sites on the bacterial photosynthetic reaction center (RC) protein were probed with pulsed electro n paramagnetic resonance (EPR spectroscopy. For these studies, Cu2+ was bou nd specifically to a surface site on native Fe2+-containing RCs from Rhodob acter sphaeroides R-26 and to the native non-heme Fe site in biochemically Fe-removed RCs, The cw and pulsed EPR results clearly indicate two spectros copically different Cu2+ environments. In the dark, the RCs with Cu2+ bound to the surface site exhibit an axially symmetric EPR spectrum with g(paral lel to) = 2.24, A(parallel to) = 160 G, g(perpendicular to) = 2.06, whereas the values g(parallel to) = 2.31, A(parallel to) = 143 G, and g(perpendicu lar to) = 2.07 were observed when Cu2+ was substituted in the Fe site. Exam ination of the light-induced spectral changes indicate that the surface Cu2 + is at least 23 iq removed from the primary donor (P+) and reduced quinone acceptor (Q(A)(-)) Electron spin-echo envelope modulation (ESEEM) spectra of these Cu-RC proteins have been obtained and provide the first direct sol ution structural information about the ligands in the surface metal site. F rom these pulsed EPR experiments, modulations were observed that are consis tent with multiple weakly hyperfine coupled N-14 nuclei in close proximity to Cu2+, indicating that two or more histidines ligate the Cu2+ at the surf ace site, Thus, metal and EPR analyses confirm that we have developed relia ble methods for stoichiometrically and specifically binding Cu2+ to a surfa ce site that is distinct from the well characterized Fe site and support th e view that Cu2+ is bound at or near the Zn site that modulates electron tr ansfer between the quinones QA and QB (Q(A)(-)Q(B) --> Q(A)Q(B)(-)) (Utschi g, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemist ry 37, 8278-8281) and proton uptake by Q(B)(-) (Paddock, M. L., Graige, M. S., Feher, G., and Okamura, hi. Y. (1999) Proc. Natl. Acad. Sci. U.S.A, 96, 6183-6188). Detailed EPR spectroscopic characterization of these Cu2+-RCs will provide a means to investigate the role of local protein environments in modulating electron and proton transfer.