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_AQ_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_AQ_B⁻.