Changes in Primary Donor Hydrogen-Bonding Interactions in Mutant Reaction Centers from Rhodobacter sphaeroides: Identification of the Vibrational Frequencies of All the Conjugated Carbonyl Groups

Tony A. Mattioli, Jo Ann C. Williams, James P. Allen, Bruno Robert

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Specific changes in the hydrogen-bonding states of the primary donor, P, in reaction centers from Rhodobacter sphaeroides bearing mutations near P were determined using near-infrared excited Fourier transform (FT) Raman spectroscopy. This technique, using 1064-nm excitation, provides the preresonantly enhanced vibrational spectrum of P in its reduced state selectively over the contributions of the other reaction center chromophores and protein and yields structural information concerning P and its hydrogen-bonding interactions. The mutations studied were as follows: Leu M160 → His, Leu L131 → His, the D9 double mutant (Leu M160 → His + Leu L131 → His), Phe M197 → His, and His L168 → Phe. These mutations were designed to introduce new, or to break existing, hydrogen bonds to the C9 and C2 carbonyl groups of P. On the basis of previous assignments [Mattioli, T. A., Hoffmann, A., Robert, B., Schrader, B., & Lutz, M. (1991) Biochemistry 30, 4648–4654], the FT Raman spectra of these mutants show the predicted changes in hydrogen bond interactions of P carbonyl groups with the protein. The results of this study have permitted us to unambiguously identify the C2 and C9 carbonyl vibrators of P in Rb. sphaeroides. The genetically introduced hydrogen bond interactions are discussed in terms of other physicochemical properties of P including the redox potential and electronic asymmetry in the P+ state. It is discussed that changes in protein hydrogen bonding to the conjugated carbonyl groups of P alone are not the sole factor that contributes to the sizable modifications of the P/P+ redox midpoint potentials, and that the chemical nature of the hydrogen bond donor plays a significant role in this modification.

Original languageEnglish
Pages (from-to)1636-1643
Number of pages8
Issue number7
Publication statusPublished - Feb 1 1994


ASJC Scopus subject areas

  • Biochemistry

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