Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB in Rhodobacter sphaeroides reaction centers

Abstract 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 − .