Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers.
Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).
Mezzetti
Alberto
A
Section de Bioénergétique, CEA Saclay, Bât 532, 91191, Gif-sur-Yvette, France.
Nabedryk
Eliane
E
Breton
Jacques
J
Okamura
Melvin Y
MY
Paddock
Mark L
ML
Giacometti
Giovanni
G
Leibl
Winfried
W
D016227:Benzoquinones / Q000737:chemistry; D004579:Electron Transport; D006873:Hydroquinones / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D016227:Benzoquinones; D006873:Hydroquinones; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; C004532:quinone