Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

摘要

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H⁺(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA⁻ and QB⁻. In this work, the pathway for H⁺(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D⁺QA⁻) and QB (DQB→D⁺QB⁻), studied by using pH-sensitive dyes. The other was the electron transfer kAB⁽¹⁾ (QA⁻QB→QAQB⁻). At pH 8.5, binding of Zn²⁺, Cd²⁺, or Ni²⁺ reduced the rates of proton uptake upon Q_A⁻ and Q_B⁻ formation as well as k_AB⁽¹⁾ by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D⁺Q_A⁻ is formed 10⁵-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn²⁺ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H⁺(2) was thus identified to be the same as for the first proton H⁺(1), close to the metal binding region Asp-H124, His-H126, and His-H128.