Electrostatic calculations of amino acid titration and electron transfer, Q-AQB--&gt;QAQ-B, in the reaction center

Beroza, Paul; Fredkin, D. R.; Okamura, M. Y.; Fehér, G.

doi:10.1016/s0006-3495(95)80406-6

Cited by 165 publications

(226 citation statements)

References 26 publications

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“…reported to have perturbed shapes (18)(19)(20)(21). We now establish that these shapes can be used as a diagnostic tool to determine the location of the active site.…”

Section: Summary and Discussionmentioning

confidence: 63%

“…This requires calculation of the electrostatic potentials, for which we employ the UHBD (16) program. We use the program HYBRID (15,17) to calculate the mean net charge as a function of pH (18)(19)(20)(21) for each ionizable group (all Lys, Arg, Asp, Glu, His, Tyr, Cys, N terminus, and C terminus) of each protein. For each residue type in a given enzyme, a plot is drawn of predicted mean net charge as a function of pH-THEMATICS.…”

Section: Methodsmentioning

confidence: 99%

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THEMATICS: A simple computational predictor of enzyme function from structure

Ondrechen

Clifton

Ringe

2001

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

215

203

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We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in proteins of known structure. Results are featured for three enzymes: triosephosphate isomerase (TIM), aldose reductase (AR), and phosphomannose isomerase (PMI). We note that TIM and AR have similar structures but catalyze different kinds of reactions, whereas TIM and PMI have different structures but catalyze similar reactions. Analysis of the theoretical microscopic titration curves for all of the ionizable residues of these proteins shows that a small fraction (3-7%) of the curves possess a flat region where the residue is partially protonated over a wide pH range. The preponderance of residues with such perturbed curves occur in the active site. Additional results are given in summary form to show the success of the method for proteins with a variety of different chemistries and structures.T here is a need to develop methods to predict protein function from structure; this need becomes particularly acute as novel protein folds are discovered for which there are no proteins of similar structure with known function. We show that theoretical microscopic titration curves (THEMATICS) can be used to identify active-site residues in enzymes of known structure. This information will prove to be important in the current challenging quest to predict protein function from sequence and structure.Knowledge of protein sequences and structures has burgeoned very recently as a result of genome sequencing (1) and structural genomics efforts. To translate such information into tangible benefits to humankind, the next step is to develop methods that enable one to predict and to establish function from structure. Techniques used to predict function from sequence or function from structure are in their infancy and rely either on analogies to related proteins of known function (2-8) or on computational searches for binding sites by docking (9) of selected sets of small molecules onto the structure. For instance, recent work has searched the Protein Data Bank (PDB; ref. 10; http:͞͞ www.rcsb.org͞pdb͞) for previously unrecognized cation-binding sites (11).However, there is as yet no reliable method to identify active sites of enzymes or other interaction sites of proteins in the absence of biochemical data, even when the structure is known. There is a wealth of biochemical data that demonstrates that active-site residues involved in specific kinds of chemistry possess predictable chemical properties that enable one to identify them as active-site residues. The most important of these properties is a perturbed pK a that can be determined experimentally by pH titrations of the activity of the enzyme. Herein we demonstrate that theoretical titration functions can identify active-site residues that are involved in Brønsted acid-base chemistry for a variety of proteins, thereby identifying the active site from the location of the residues.In an experimental titration curve, one typically plots pH as a function of the volume of...

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“…reported to have perturbed shapes (18)(19)(20)(21). We now establish that these shapes can be used as a diagnostic tool to determine the location of the active site.…”

Section: Summary and Discussionmentioning

confidence: 63%

Section: Methodsmentioning

confidence: 99%

THEMATICS: A simple computational predictor of enzyme function from structure

Ondrechen

Clifton

Ringe

2001

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

215

203

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“…The x-ray structures of the Wt RCs show close proximity (<5 A) between GluH73, AspL213, and ArgL217 (Fig. 1), and calculations (13,37) show both carboxylic acid residues to be partially ionized over a broad pH range. We suggest that mutation of AspL213 to a neutral species removes a (partial) negative charge close to QB, thereby stabilizing the QB anion.…”

Section: Discussionmentioning

confidence: 91%

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

Takahashi

Wraight

1996

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

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The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M-and H-subunit residues also close by. Studies on RCs altered by site-directed mutagenesis strongly implicate two ionizable residues in functional proton transfer to the QB binding site in Rb. sphaeroides-Glu and Asp at positions 212 and 213, respectively, of the L subunit (6-9).These are the two ionizable residues nearest to the QB head group (Fig. 1). Alteration of AspL213 to Asn had severe effects, including a-very large increase in the QAQB = QAQB equilibrium in favor of QB reduction and modification of the pH dependence of the equilibrium, especially at acid pH (7-9).Also, transfer of the second electron to QB was almost completely blocked, due to failure in the transfer of the first proton necessary for the double reduction of QB: QAQB + H+X--QAQBH-. Alteration of GluL212 to Gln also had significant effects on the quinone reactions (6,8

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“…(14,16,60,61). Thus, the L210DN/M17DN double mutant should be at least as hospitable, and a preference for the anion species is consistent with this.…”

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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Electrostatic calculations of amino acid titration and electron transfer, Q-AQB-->QAQ-B, in the reaction center

Cited by 165 publications

References 26 publications

THEMATICS: A simple computational predictor of enzyme function from structure

THEMATICS: A simple computational predictor of enzyme function from structure

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

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

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