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Protein-Cofactor Interactions in Bacterial Reaction Centers from Rhodobacter sphaeroides R-26: II. Geometry of the Hydrogen Bonds to the Primary Quinone by ¹H and ²H ENDOR Spectroscopy

M. Flores ^* , R. Isaacson ^*, E. Abresch ^*, R. Calvo ^* , W. Lubitz  and G. Feher ^*

^* Department of Physics, University of California at San Diego, La Jolla, California; Max-Planck Institut für Bioanorganische Chemie, Mülheim an der Ruhr, Germany; and Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas and INTEC, Universidad Nacional del Litoral and CONICET, Santa Fe, Argentina

Correspondence: Address reprint requests to G. Feher, Tel.: 858-534-4388; E-mail: gfeher{at}ucsd.edu.

The geometry of the hydrogen bonds to the two carbonyl oxygens of the semiquinone in the reaction center (RC) from the photosynthetic purple bacterium Rhodobacter sphaeroides R-26 were determined by fitting a spin Hamiltonian to the data derived from ¹H and ²H ENDOR spectroscopies at 35 GHz and 80 K. The experiments were performed on RCs in which the native Fe²⁺ (high spin) was replaced by diamagnetic Zn²⁺ to prevent spectral line broadening of the due to magnetic coupling with the iron. The principal components of the hyperfine coupling and nuclear quadrupolar coupling tensors of the hydrogen-bonded protons (deuterons) and their principal directions with respect to the quinone axes were obtained by spectral simulations of ENDOR spectra at different magnetic fields on frozen solutions of deuterated in H₂O buffer and protonated in D₂O buffer. Hydrogen-bond lengths were obtained from the nuclear quadrupolar couplings. The two hydrogen bonds were found to be nonequivalent, having different directions and different bond lengths. The H-bond lengths r_OH are 1.73 ± 0.03 Å and 1.60 ± 0.04 Å, from the carbonyl oxygens O₁ and O₄ to the NH group of Ala M260 and the imidazole nitrogen N of His M219, respectively. The asymmetric hydrogen bonds of affect the spin density distribution in the quinone radical and its electronic structure. It is proposed that the H-bonds play an important role in defining the physical properties of the primary quinone, which affect the electron transfer processes in the RC.