EPR investigation of Cu{sup 2+}-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, Cu{sup 2+} was bound specifically to a surface site on native Fe{sup 2+}-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 Cu{sup 2+} environments. In the dark, the RCs with Cu{sup 2+} bound to the surface site exhibit an axially symmetric EPR spectrum with g{sub {parallel}} = 2.24, A{sub {parallel}} = 160 G, g{sub {perpendicular}} = 2.06, whereas the values g{sub {parallel}} = 2.31, A{sub {parallel}} = 143 G, and g{sub {perpendicular}} = 2.07 were observed when Cu{sup 2+} was substituted in the Fe site. Examination of the light-induced spectral changes indicate that the surface Cu{sup 2+} is at least 23 {angstrom} removed from the primary donor (P{sup +}) and reduced quinone acceptor (Q{sub A}{sup -}). 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 {sup 14}N nuclei in close proximity to Cu{sup 2+}, indicating that two or more histidines ligate the Cu{sup 2+} at the surface site. Thus, metal and EPR analyses confirm that we have developed reliable methods for stoichiometrically and specifically binding Cu{sup 2+} to a surface site that is distinct from the well characterized Fe site and support the view that Cu{sup 2+} is bound at or near the Zn site that modulates electron transfer between the quinones Q{sub A} and Q{sub B} (Q{sub A}{sup -}Q{sub B} {yields} Q{sub A}Q{sub B}{sup -}) (Utschig, L. M., Ohigashi, Y., Thurnauer, M. C., and Tiede, D. M (1998) Biochemistry 37, 8278-8281) and proton uptake by Q{sub B}{sup -} (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 Cu{sup 2+}-RCs will provide a means to investigate the role of local protein environments in modulating electron and proton transfer.
- Research Organization:
- Argonne National Laboratory (ANL)
- Sponsoring Organization:
- SC
- DOE Contract Number:
- AC02-06CH11357
- OSTI ID:
- 942665
- Report Number(s):
- ANL/CHM/JA-33619
- Journal Information:
- Biochemistry, Journal Name: Biochemistry Journal Issue: 11 ; 2000 Vol. 39; ISSN 0006-2979
- Country of Publication:
- United States
- Language:
- ENGLISH
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