Identification of the proton pathway in bacterial reaction centers:: Inhibition of proton transfer by binding of Zn2+ or Cd2+

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(B)H)(-), where Q(A) is the primary quinone acceptor), Zn2+ and Cd2+ bound stoichiometrically to the RC (KD less than or equal to 0.5 mu M) 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 kAB(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 (greater than or equal to 10(2)-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D(+.)Q(A)Q(B)(-.) --> DQ(A)Q(B) suggests that the binding site is located far (> 10 Angstrom) 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.