Small Weak Acids Reactivate Proton Transfer in Reaction Centers from Rhodobacter sphaeroides Mutated at AspL210 and AspM17

Abstract

In reaction centers of Rhodobacter sphaeroides, site-directed mutagenesis has implicated several acidic residues in the delivery of protons to the secondary quinone (Q B ) during reduction to quinol. In a double mutant (Asp L210 3 Asn ؉ Asp M17 3 Asn) that is severely impaired in proton transfer capability over a wide pH range, proton transfer was "rescued" by added weak acids. For low pK a acids the total concentration of salt required near neutral pH was high. The ionic strength effect of added salts stimulated the rate of proton-coupled electron transfer at pH < 7, but decreased it at pH > 7.5, indicating an effective isoelectric point between these limits. In this region, a substantial rate enhancement by weak acids was clearly evident. A Brønsted plot of activity versus pK a of the rescuing acids was linear, with a slope of ؊1, and extrapolated to a diffusion-limited rate at pK a app ≈ 1. However, the maximum rate at saturating concentrations of acid did not correlate with pK a , indicating that the acid and anion species compete for binding, both with weak affinity. This model predicts that pK a app corresponds to a true pK a ‫؍‬ 4 -5, similar to that for a carboxylic acid or Q B ؊ , itself.Only rather small, neutral acids were active, indicating a need to access a small internal volume, suggested to be a proton channel to the Q B domain. However, the on-rates were near the diffusion limit. The implications for intraprotein proton transfer pathway design are discussed.Light absorption by photosynthetic reaction centers (RCs) 2 drives the formation of an electrochemical gradient of H ϩ ions (protons) across the coupling membrane, the thylakoid membrane in chloroplasts and cell membrane in bacteria, and the generation of mobile reducing power. The transfer of electrons in the primary photochemical events generates most of the electrical component, whereas electron-coupled proton uptake and release accompanying the redox reactions of secondary donors and acceptors is largely responsible for the proton concentration gradient (⌬pH) (1).In Rhodobacter sphaeroides, reducing equivalents are stored in the double reduction of the secondary ubiquinone, Q B , via the primary quinone, Q A , and quinol is released into the membrane after two lightactivated turnovers of the RC (2, 3). Each turnover results in transfer of an electron to the quinones from the primary donor, P, a special pair of bacteriochlorophylls (4 -7). The oxidized primary donor, P ϩ , is rereduced by a secondary donor after each photoactivation, and the events in the acceptor quinone complex can be summarized as (8 -10) in Scheme 1. The proton stoichiometric factors, a, b, etc., indicate the variable influence that the different quinone states have on nearby ionizable amino acid residues (10 -14). The RC quinones are well buried in the protein, and proton transfer to Q B , which accompanies the second electron transfer to form Q B H 2 , must extend over a distance of 13-15 Å. The delivery pathway has been partially mapped out by site-directed mutagen...

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