Characterization of second site mutations show that fast proton transfer to QB- is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

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Abstract

The structural basis for proton coupled electron transfer to QB in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 → Asp, Arg M233 → Cys, Arg H177 → His) that complement the effects of the deleterious Asp L213 → Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate kAB(2)(QA-QB- + H+ → QAQBH-) by at least 10³ (pH 7.5) and a recombination rate kBD (D+QAQB- → DQAQB) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of kAB(2) on the free energy for electron transfer (δΔGet). kAB(2) was independent of δΔGet in DN(L213) RCs, but dependent on δΔGet in native and all suppressor RCs. This shows that proton transfer limits the kAB(2) reaction with a rate of 0.1s-1 in DN(L213) RCs but is not rate limiting and at least 108-fold faster in native and 105-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near QB-; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near QB.

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