Abstract
The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, Q(B). This process involves the transfer of two protons from the aqueous solution to the protein-bound Q(B) molecule. The second proton, H(+)(2), is supplied to Q(B) by Glu-L212, an internal residue protonated in response to formation of Q(A)(-) and Q(B)(-). 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 Q(A) (DQ(A)-->D(+)Q(A)(-)) and Q(B) (DQ(B)-->D(+)Q(B)(-)), studied by using pH-sensitive dyes. The other was the electron transfer k(AB)((1)) (Q(A)(-)Q(B)-->Q(A)Q(B)(-)). At pH 8.5, binding of Zn(2+), Cd(2+), or Ni(2+) reduced the rates of proton uptake upon Q(A)(-) and Q(B)(-) formation as well as k(AB)((1)) by approximately an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D(+)Q(A)(-) is formed 10(5)-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 Zn(2+) 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.