Electrostatic influence of Q(A) reduction on the IR vibrational mode of the 10a-ester C=O of H(A) demonstrated by mutations at residues Glu l104 and Trp L100 in reaction centers from Rhodobacter sphaeroides

Jacques Breton, Eliane Nabedryk, James Allen, Joann Williams

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The light-induced Q(A)-/Q(A) FTIR difference spectrum of the photoreduction of the primary quinone Q(A) in reaction centers (RC(s)) from Rhodobacter sphaeroides exhibits a set of complex differential bands between 1750 and 1715 cm-1. Several of these features correspond in frequency to bands that bleach in the H(A)-/H(A) FTIR difference spectra of the photoreduction of the bacteriopheophytin electron acceptor (H(A). Since the 10a-ester C=O from HA and the side chains of protonated carboxylic acids would be expected to contribute in this spectral region, mutations were designed at Trp L100 and Glu L104, which have been proposed to form hydrogen bonds to the 10a-ester and the 9-keto carbonyls on ring V of H(A), respectively. The Q(A)-/Q(A) spectra measured in H2O and 2H2O of RC(s) from wild type (WT) were compared to those of RCs with the mutation Trp to Phe at L100 [WF(L100)], Glu to Leu at L104 [EL(L104)], or both mutations [EL(L104)/WF(L100)]. The spectra of the mutants in the 18001400 cm-1 frequency range exhibit only limited perturbations compared to those of WT, indicating the absence of significant structural changes due to the mutations. Part of a differential signal centered around 1732 cm-1 in the spectrum of WT RCs is downshifted by ≃7 cm-1 in EL(L104), while it is upshifted by 11 cm-1 in WF(L 100). This upshift of the differential signal is assigned to the frequency change of the 10a-ester C=O of HA induced by the rupture of the hydrogen bond with Trp L100. The 1H2O-minus-2H2O double- difference spectrum of WT RCs exhibits a characteristic differential signal positive at 1730 cm-1 and negative at 1724 cm-1 that is absent in the corresponding spectra of EL(L104) and of the double mutant, implicating Glu L104 in the Q(A)-/Q(A) spectral changes. This differential signal is strongly modified in frequency and amplitude in the 1H2O-minus-2H2O spectrum of WF(L100), indicating that it does not correspond to a direct response of the C=O mode of the Glu L104 side chain upon Q(A) reduction. Instead, perturbation of the hydrogen bond of the 9-keto C=O with Glu L104 is proposed to induce a change of electron density on ring V of H(A), thereby altering the frequency of the 10a-ester C=O that is in partial conjugation with ring V. The loss of the hydrogen bond to the 9-keto C=O of HA due to the Glu L 104 to Leu mutation or the alteration of the strength of the hydrogen bond by 1H/2H exchange on Glu L104 appears to produce such effects. Thus, the Q(A)- /Q(A) spectra above 1700 cm-1 are dominated by contributions from the 10a- ester C=O of H(A), with most of the differential signals assigned to a small frequency downshift of the 10a-ester C=O of H(A) in response to Q(A) reduction. The complexity of the signals implies a structural heterogeneity of the conformation and hydrogen bonding of the 10a-ester C=O of H(A), which may be related to the functional heterogeneity observed in electron transfer kinetics. The present FT1R results show that the reduction of Q(A) can induce a pronounced electrostatic effect on molecular vibrations of chemical groups located about 109 away from Q(A). They also demonstrate that, within experimental limits, the proton uptake observed at pH 7 upon Q(A) photoreduction [McPherson, P. H., Okamura, M. Y., and Feher, G. (1988) Biochim. Biophys. Acta 934, 348-368] involves none of the exchangeable carboxylic groups of the RC.

Original languageEnglish (US)
Pages (from-to)4515-4525
Number of pages11
Issue number15
StatePublished - Apr 15 1997

ASJC Scopus subject areas

  • Biochemistry

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