Identification of the proton pathway in bacterial reaction centers: replacement of Asp-M17 and Asp-L210 with asn reduces the proton transfer rate in the presence of Cd2+

Abstract

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the reduction and protonation of a bound quinone molecule Q(B) (the secondary quinone electron acceptor). We investigated the proton transfer pathway by measuring the proton-coupled electron transfer, k(AB)((2)) [Q(A)Q(B) + H(+) --> Q(A)(Q(B)H)(-)] in native and mutant RCs in the absence and presence of Cd(2+). Previous work has shown that the binding of Cd(2+) decreases k(AB)((2)) in native RCs approximately 100-fold. The preceding paper shows that bound Cd(2+) binds to Asp-H124, His-H126, and His-H128. This region represents the entry point for protons. In this work we investigated the proton transfer pathway connecting the entry point with Q(B) by searching for mutations that greatly affect k(AB)((2)) ( greater, similar10-fold) in the presence of Cd(2+), where k(AB)((2)) is limited by the proton transfer rate (k(H)). Upon mutation of Asp-L210 or Asp-M17 to Asn, k(H) decreased from approximately 60 s(-1) to approximately 7 s(-1), which shows the important role that Asp-L210 and Asp-M17 play in the proton transfer chain. By comparing the rate of proton transfer in the mutants (k(H) approximately 7 s(-1)) with that in native RCs in the absence of Cd(2+) (k(H) >/= 10(4) s(-1)), we conclude that alternate proton transfer pathways, which have been postulated, are at least 10(3)-fold less effective.

Figure 1

Absorbance decay of the semiquinones at 450 nm as a function of time after the second laser flash in the presence of 10 μM, 100 μM, or 1000 μM CdSO₄ in the DN(M17) [Asp-M17 → Asn] RCs. From the decay, the rate constant k_AB⁽²⁾ was determined. Note the slowing of the kinetics with increasing CdSO₄ concentrations. A similar behavior is observed in the DN(L210) [Asp-L210 → Asn] RCs (data not shown). This is in contrast to the behavior of native RCs, where no change in the kinetics is observed above CdSO₄ concentrations of 10 μM. The pedestal at long times is due to the absorbance change of Cyt c used to reduce the primary donor. Conditions: 2 μM RCs in TL buffer, pH 7.7; concentrations of CdSO₄ as indicated.

Figure 2

The slow phase of k_AB⁽²⁾ as a function of CdSO₄ concentration for native (●), and mutant DN(M17) (■) and DN(L210) (○) RCs. In native RCs, k_AB⁽²⁾ is independent of CdSO₄ concentration above 10 μM. In mutant RCs, k_AB⁽²⁾ displays a sigmoidal dependence on CdSO₄ concentration. The solid curve represents a theoretical fit using the kinetic model described by Eq. 5 (see also Eq. A4 of Appendix) with the parameters shown in Table 2. The drastic difference in behavior between native and mutant RCs is because of the lack of competition of k_off with k_AB⁽²⁾(Cd²⁺) in native RCs (see Discussion). Conditions: same as in Fig. 1.

Figure 3

The absorbance decay of the semiquinones at 450 nm as a function of time in native and the mutant DN(M17) and DN(L210) RCs in the presence of 1 mM CdSO₄. This represents the rate of proton transfer in RCs with a bound Cd²⁺ metal ion. Note that k_AB⁽²⁾ is decreased ≈10-fold in the mutant RCs compared with native RCs. The observed decay rate for the two mutant RCs is the same within experimental error. Conditions: same as in Fig. 1.

Figure 4

Part of the RC structure with a bound Cd²⁺ showing the region between the bound metal ion and Q_B⨪ [modified from Axelrod et al. (25)]. The metal (large sphere) is coordinated (solid lines) to Asp-H124, His-H126, His-H128, and three water molecules. Located in this region are Asp-L210, Asp-M17, Asp-L213, Ser-L223 (as indicated), and several water molecules (small spheres). The kinetic results presented in this work show that Asp-L210 and Asp-M17 are involved in proton transfer to Q_B⨪ in the presence of a bound Cd²⁺. The most likely proton transfer pathways connecting the surface near Cd²⁺ to Q_B⨪ are indicated by the dashed lines. In the absence of a bound Cd²⁺, the same pathways are expected to predominate with an even greater effectiveness.