Conformational heterogeneity of the bacteriopheophytin electron acceptor HA in reaction centers from Rhodopseudomonas viridis revealed by Fourier transform infrared spectroscopy and site-directed mutagenesis.
The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.
Breton
J
J
Section de Bioénergétique, Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, Gif-sur-Yvette, France. cadara3@dsvidf.cea.fr
Bibikova
M
M
Oesterhelt
D
D
Nabedryk
E
E
D019943:Amino Acid Substitution / Q000235:genetics; D016227:Benzoquinones / Q000737:chemistry; D017666:Deuterium Oxide; D004579:Electron Transport; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000235:genetics; D010674:Pheophytins / Q000737:chemistry* / Q000235:genetics; D010777:Photochemistry; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011522:Protons; D012241:Rhodopseudomonas / Q000737:chemistry* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D055672:Static Electricity; D014364:Tryptophan / Q000235:genetics
D016227:Benzoquinones; D010674:Pheophytins; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D005973:Glutamine; C006678:bacteriopheophytin; D018698:Glutamic Acid; C004532:quinone; D010649:Phenylalanine; D014364:Tryptophan; D017666:Deuterium Oxide