Electrostatic influence of QA reduction on the IR vibrational mode of the 10a-ester C==O of HA demonstrated by mutations at residues Glu L104 and Trp L100 in reaction centers from Rhodobacter sphaeroides.
The light-induced QA-/QA FTIR difference spectrum of the photoreduction of the primary quinone (QA) in reaction centers (RCs) 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 HA-/HA FTIR difference spectra of the photoreduction of the bacteriopheophytin electron acceptor (HA). 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 HA, respectively. The QA/-/QA spectra measured in IH2O and 2H2O of RCs 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 1800-1400 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 approximately 7 cm(-1) in EL(L104), while it is upshifted by approximately 11 cm(-1) in WF(L100). 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 QA-/QA 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 QA 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 HA, 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 L104 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 QA-/QA spectra above 1700 cm-1 are dominated by contributions from the 10a-ester C==O of HA, with most of the differential signals assigned to a small frequency downshift of the 10a-ester C==O of HA in response to QA reduction. The complexity of the signals implies a structural heterogeneity of the conformation and hydrogen bonding of the 10a-ester C==O of HA, which may be related to the functional heterogeneity observed in electron transfer kinetics. The present FTIR results show that the reduction of QA can induce a pronounced electrostatic effect on molecular vibrations of chemical groups located about 10 A away from QA. They also demonstrate that, within experimental limits, the proton uptake observed at pH 7 upon QA photoreduction [McPherson, P. H., Okamura, M. Y., & Feher, G. (1988) Biochim. Biophys. Acta 934, 348-368] involves none of the exchangeable carboxylic groups of the RC.
Breton
J
J
Département de Biologie Cellulaire et Moléculaire, CEA-Saclay, Gif-sur-Yvette, France.
Nabedryk
E
E
Allen
J P
JP
Williams
J C
JC
D000595:Amino Acid Sequence; D004563:Electrochemistry; D004952:Esters; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics*; D006860:Hydrogen Bonding; D009154:Mutation; D010674:Pheophytins / Q000737:chemistry / Q000378:metabolism*; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics*; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics*
D004952:Esters; D010674:Pheophytins; D045322:Photosynthetic Reaction Center Complex Proteins; D011809:Quinones; C006678:bacteriopheophytin; D018698:Glutamic Acid; D014364:Tryptophan