Mechanism of Proton-Coupled Electron Transfer for Quinone (QB) Reduction in Reaction Centers of Rb. Sphaeroides

Graige, M. S.; Paddock, Mark L.; Bruce, J. Malcolm; Fehér, G.; Okamura, M. Y.

doi:10.1021/ja960056m

Cited by 197 publications

(242 citation statements)

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“…A detailed procedure for reconstitution of the Q A binding site with naphthoquinone and Q B binding site with ubiquinone has been presented (11). The occupancy of the Q A and Q B sites after reconstitution were determined from the amplitudes of transients associated with charge recombination, k AD (18).…”

Section: Methodsmentioning

confidence: 99%

“…If k C Ͼ k ET , the reaction is rate-limited by electron transfer. To determine which of these two cases prevails, a driving force assay was used (11). In this assay, quinones with different redox potentials (i.e., driving force) are substituted for Q A , thereby changing the driving force for electron transfer and, thus, the intrinsic rate constant, k ET .…”

mentioning

confidence: 99%

“…Naphthoquinones (NQ) with redox potentials different from that of native Q 10 were substituted into the Q A site whereas Q 10 was retained in the Q B site. (11)(12)(13). The substitution of Q A changes the intrinsic rate of electron transfer, k ET , without affecting k C since the Q B site is unchanged and the structures of the substituted Q A molecules are nearly identical.…”

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confidence: 99%

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Conformational gating of the electron transfer reaction Q _A ⁻ ^⋅ Q _B → Q _A Q _B ⁻ ^⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Graige

Fehér

Okamura

1998

Proc. Natl. Acad. Sci. U.S.A.

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The mechanism of the electron transfer reaction, Q A ؊ ⅐ Q B 3 Q A Q B ؊ ⅐ , was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q 10 in the Q A binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from Q A ؊ ⅐ to Q B was measured by three independent methods: a functional assay involving cytochrome c 2 to measure the rate of Q A ؊ ⅐ oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from Q A ؊ ⅐ to Q B does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of Q B by Ϸ5 Å as observed in the x-ray studies of Stowell et al.

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Section: Methodsmentioning

confidence: 99%

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Conformational gating of the electron transfer reaction Q _A ⁻ ^⋅ Q _B → Q _A Q _B ⁻ ^⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Graige

Fehér

Okamura

1998

Proc. Natl. Acad. Sci. U.S.A.

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211

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“…For example, fluoride (pK a ϭ 3.16) and phosphate (pK a ϭ 2.15) were unable to fully restore the rate, or even to attain the same level as azide (pK a ϭ 4.72). However, this is readily accounted for if we allow for the anion (A Ϫ ) to interact at the same site as the acid (57,58) and is largely unaffected by conditions and mutations that induce significant electrostatic changes in the acid cluster of the Q B domain (28,30). We therefore take the wild-type value of k ET ϭ 10 6 s Ϫ1 .…”

Section: L210mentioning

confidence: 99%

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

Takahashi

Wraight

2006

Journal of Biological Chemistry

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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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“…A proton-activated electron-transfer mechanism has been proposed which suggests the fast protonation of Q B , prior to the slower rate-limiting electron-transfer (6,8,13). However, in chromatophores from R. sphaeroides, it was shown that, at the first flash, Q B can be protonated with a pK of ∼6.8 (14), suggesting that there might be no fundamental differences between the first and second electron-transfer processes.…”

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Effect of Binding of Cd²⁺ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

et al. 2002

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In bacterial reaction center of Rhodobacter sphaeroides, Cd 2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (k AB (1)) and second flash (k AB (2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogenbond networks involving water molecules. We show that (1) the combined effects of Cd 2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd 2+ ] titration curves of k AB (2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S. A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd 2+ binds; (2) in the L209PY mutant, the pH titration curves of k AB (1), k AB (2), and k H + , the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 µM Cd 2+

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Mechanism of Proton-Coupled Electron Transfer for Quinone (Q_B) Reduction in Reaction Centers of Rb. Sphaeroides

Abstract: The mechanism of the proton-coupled electron transfer reaction, Q A -Q B -

Cited by 197 publications

References 66 publications

Conformational gating of the electron transfer reaction Q _A ⁻ ^⋅ Q _B → Q _A Q _B ⁻ ^⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Conformational gating of the electron transfer reaction Q _A ⁻ ^⋅ Q _B → Q _A Q _B ⁻ ^⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

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

Effect of Binding of Cd²⁺ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

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Mechanism of Proton-Coupled Electron Transfer for Quinone (QB) Reduction in Reaction Centers of Rb. Sphaeroides

Abstract: The mechanism of the proton-coupled electron transfer reaction, Q A -Q B -

Cited by 197 publications

References 66 publications

Conformational gating of the electron transfer reaction Q A − ⋅ Q B → Q A Q B − ⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Conformational gating of the electron transfer reaction Q A − ⋅ Q B → Q A Q B − ⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

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

Effect of Binding of Cd2+ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways

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Mechanism of Proton-Coupled Electron Transfer for Quinone (Q_B) Reduction in Reaction Centers of Rb. Sphaeroides

Conformational gating of the electron transfer reaction Q _A ⁻ ^⋅ Q _B → Q _A Q _B ⁻ ^⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Conformational gating of the electron transfer reaction Q _A ⁻ ^⋅ Q _B → Q _A Q _B ⁻ ^⋅ in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

Effect of Binding of Cd²⁺ on Bacterial Reaction Center Mutants: Proton-Transfer Uses Interdependent Pathways