Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB 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)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 hake been replaced with Asn. Mutation of both residues decreases drastically k(AB), 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)(-). (C) 2002 Elsevier Science B.V. All rights reserved.