Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn 2+ or Cd 2+

Abstract

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule Q_B (the secondary quinone acceptor). A unique pathway for proton transfer to the Q_B site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer k_AB⁽²⁾ (Q_A^−•Q_B^−• + H⁺ → Q_A(Q_BH)⁻, where Q_A is the primary quinone acceptor). Zn²⁺ and Cd²⁺ bound stoichiometrically to the RC (K_D ≤ 0.5 μM) and reduced the observed value of k_AB⁽²⁾ 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the k_AB⁽²⁾ reaction. In native RCs, k_AB⁽²⁾ was previously shown to be rate-limited by electron transfer based on the dependence of k_AB⁽²⁾ on the driving force for electron transfer. Upon addition of Zn²⁺ or Cd²⁺, k_AB⁽²⁾ became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 10²-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D^+•Q_AQ_B^−• → DQ_AQ_B suggests that the binding site is located far (>10 Å) from Q_B. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to Q_B^−• occurs.