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 electron paramagnetic resonance (EPR) spectroscopy. For these studies, Cu2+ was bound specifically to a surface site on native Fe2+-containing RCs from Rhodobacter 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 spectroscopically different Cu2+ environments. In the dark, the RCs with Cu2+ bound to the surface site exhibit an axially symmetric EPR spectrum with g(parallel) = 2.24, A(parallel) = 160 G, g(perpendicular) = 2.06, whereas the values g(parallel) = 2.31, A(parallel) = 143 G, and g(perpendicular) = 2.07 were observed when Cu(2+) was substituted in the Fe site. Examination of the light-induced spectral changes indicate that the surface Cu2+ is at least 23 A removed from the primary donor (P+) and reduced quinone acceptor (QA-). Electron spin-echo envelope modulation (ESEEM) spectra of these Cu-RC proteins have been obtained and provide the first direct solution structural information about the ligands in the surface metal site. From these pulsed EPR experiments, modulations were observed that are consistent with multiple weakly hyperfine coupled 14N nuclei in close proximity to Cu2+, indicating that two or more histidines ligate the Cu2+ at the surface site. Thus, metal and EPR analyses confirm that we have developed reliable methods for stoichiometrically and specifically binding Cu2+ to a surface site that is distinct from the well characterized Fe site and support the view that Cu2+ is bound at or near the Zn site that modulates electron transfer between the quinones QA and QB (QA-QB --> QAQB-) (Utschig, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemistry 37, 8278-8281) and proton uptake by QB- (Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. 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.
Utschig
L M
LM
Chemistry Division, Argonne National Laboratory, Argonne, Illinois 60439, USA. utschig@anl.gov
Poluektov
O
O
Tiede
D M
DM
Thurnauer
M C
MC
D001665:Binding Sites; D003300:Copper / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods; D004579:Electron Transport; D006639:Histidine / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008094:Lithium / Q000737:chemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides; D013861:Thiocyanates / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
D008024:Ligands; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D013861:Thiocyanates; D006639:Histidine; D003300:Copper; D008094:Lithium; D007501:Iron; D015032:Zinc