The Characterization of the QA Binding Site of the Reaction Center of Rhodopseudomonas sphaeroides

Gunner, M. R.; Braun, B.; Bruce, J. Malcolm; Dutton, P. Leslie

doi:10.1007/978-3-642-82688-7_41

Cited by 23 publications

(19 citation statements)

References 9 publications

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“…These results are at least qualitatively similar to those found for the QA site (8). Work with the QA site has further demonstrated not only that there is no requirement for a para-carbonyl structure but also that the contribution to binding free energy from the "second" carbonyl is small (8).…”

Section: Discussionsupporting

confidence: 82%

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In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.

Giangiacomo

Dutton

1989

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

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The secondary quinone-binding site (QB site) of bacterial reaction centers from Rhodobacter sphaeroides is generally regarded to be highly specific for its native ubiquinone-10 molecule. We demonstrate here that this is a misconception rooted in the kinetic methods used to assay for occupancy of a quinone in the QB site. We show that observance of occupancy of the QB site, revealed by kinetic assay, is sensitive to the free-energy difference for electron transfer between the quinone at the primary quinone-binding site (QA site) and the QB site (-AG0 ). For many of the compounds previously tested for binding at the QB site, the -AGO. between QA and QB is too small to permit detection of the functional quinone in the QB site. With an increased -AGO. achieved by replacing the native ubiquinone-10 at the QA site with lowerpotential quinones or by testing higher-potential QB candidates, it is shown that the QB site binds and functions with the unsubstituted 1,4-benzoquinone, 1,4-naphthoquinone, and 9,10-phenanthraquinone, as well as with their various substituted forms. Moreover, quinones with the ortho-carbonyl configuration appear to function in a similar manner to quinones with the para-carbonyl configuration.The photochemical reaction center of photosynthetic bacteria is an integral membrane protein that contains four bacteriochlorophylls (BChls), two bacteriopheophytins (BPhs), and two quinones associated with discrete catalytic sites, designated the primary and secondary quinone-binding sites (QA and QB, respectively; for review, see ref. 1). After light excitation of a special pair of bacteriochlorophylls (BChl2), an electron is transferred by way of BPh to the quinone at the QA site to form the semiquinone (QA) and the state BChl'-QQB, where BChl' is the oxidized cation radical of BChl2 (2). The QA then reduces the quinone bound at the QB site to form BChl'QAQB (3,4) In the bacterial reaction centers from Rhodobacter sphaeroides the native ubiquinone-10 (UQ-10) molecules can be reversibly removed from both the QA and QB sites (5, 6).Moreover, the QA site can be functionally reconstituted with a wide variety of other natural and synthetic quinone structures (6-8). In contrast, with the exception of one observation (9), parallel efforts to reconstitute QB activity have been successful only with quinones containing the native ubiquinone (UQ) configuration (10-13). These negative findings have led to the sentiment that the QB site, unlike the QA site, is designed to provide stringent binding requirements for the UQ structure (10,12,13). However, such a view is not entirely consistent with the well-known, broad specificity of the QB site for a variety of herbicide structures (14-16). These apparently unusual characteristics have prompted us to enquire further into the specificity of the QB site for quinone structures. We have questioned in particular the methods of assay used to establish the binding strength of a quinone to the QB site and have drawn upon the detailed general description for QB function t...

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Section: Discussionsupporting

confidence: 82%

“…Moreover, the QA site can be functionally reconstituted with a wide variety of other natural and synthetic quinone structures (6)(7)(8). In contrast, with the exception of one observation (9), parallel efforts to reconstitute QB activity have been successful only with quinones containing the native ubiquinone (UQ) configuration (10)(11)(12)(13).…”

mentioning

confidence: 99%

In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.

Giangiacomo

Dutton

1989

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

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“…Dissociation constants of quinones for the QA site of the RC protein diluted from the preparations to assay concentrations of 0.05-0.2 ,uM in the hexane and aqueous systems were determined from functional QA site occupancy versus concentration of added quinone (T = 295 ± 2 K) (19,20). The standard free energy of binding (molar standard state) was obtained from the measured Kd by AGB = RTlnKd.…”

Section: Solvation Analysismentioning

confidence: 99%

“…The standard free energy of binding (molar standard state) was obtained from the measured Kd by AGB = RTlnKd. The Kd values of the nonfunctional compounds were determined by competition with an active quinone (19 is comparable with the width of the xenon flash.…”

Section: Solvation Analysismentioning

confidence: 99%

“…Binding constants are measured by a sensitive photochemical assay that addresses functional quinone occupancy on a nanosecond time scale. Thus, the method obviates interference from diffusional encounter and allows measurements at very low protein concentrations (19,20). In addition to these features, the x-ray crystallographic structures of the RC proteins from Rb.…”

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confidence: 99%

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Experimental resolution of the free energies of aqueous solvation contributions to ligand-protein binding: quinone-QA site interactions in the photosynthetic reaction center protein.

Warncke

Dutton

1993

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

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Equilibrium binding free energies of 14 benzo-, naphtho-, and anthraquinone cofactors have been determined at the QA redox catalytic site of the purified photosynthetic reaction center protein from Rhodobacter sphaeroides solubilized in water (AGB,w), in hexane solution containing 30 mM water (AGTB,hh), and after partial dehydration (AGTB,dh) (1-4). A small organic molecule, whether a cofactor, substrate, or inhibitor, gains a strong favorable contribution from the hydrophobic effect (5-7) and encounters unfavorable contributions from loss of dipolar and hydrogen bond (2, 4) contacts with the solvent upon transfer from the bulk aqueous phase into the protein interior binding site. Upon entry to the site, the molecule fits into the contours and establishes new interactions, the description of which is essential to understanding molecular recognition and catalysis. Moreover, the ligand may again encounter water that may require relocation before the binding is complete (8-12). The important contribution of protein hydration water to the thermodynamics and specificity ofligand-protein binding has been discussed (e.g., see refs. 3 and 4) and investigated by computational methods (13). Quantitative descriptions of the role of water based on experimental approaches, however, remain a challenge.In this report, we address the free energies of aqueous solvation contributions to ligand-protein affinities by comparing binding free energies determined in water with those determined in hexane solution. The reaction center (RC) protein from Rhodobacter sphaeroides (14, 15) is well-suited for this type of analysis, because it can be solubiized in water, hexane, and partially dehydrated hexane solutions (16-18). We focus on the equilibrium binding interaction between the QA redox catalytic site of the RC protein and a series of 14 replacement quinone cofactors and 8 selected structural analogues. Binding constants are measured by a sensitive photochemical assay that addresses functional quinone occupancy on a nanosecond time scale. Thus, the method obviates interference from diffusional encounter and allows measurements at very low protein concentrations (19,20). In addition to these features, the x-ray crystallographic structures of the RC proteins from Rb. sphaeroides (21,22) and Rhodopseudomonas viridis (23)

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Mechanism of initial rapid rate retardation in cellobiohydrolase catalyzed cellulose hydrolysis

Jalak

Väljamaë

2010

Biotech & Bioengineering

134

157

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Despite intensive research, the mechanism of the rapid retardation in the rates of cellobiohydrolase (CBH) catalyzed cellulose hydrolysis is still not clear. Interpretation of the hydrolysis data has been complicated by the inability to measure the catalytic constants for CBH-s acting on cellulose. We developed a method for measuring the observed catalytic constant (k(obs)) for CBH catalyzed cellulose hydrolysis. It relies on in situ measurement of the concentration of CBH with the active site occupied by the cellulose chain. For that we followed the specific inhibition of the hydrolysis of para-nitrophenyl-beta-D-lactoside by cellulose. The method was applied to CBH-s TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium and their isolated catalytic domains. Bacterial microcrystalline cellulose, Avicel, amorphous cellulose, and lignocellulose were used as substrates. A rapid decrease of k(obs) in time was observed on all substrates. The k(obs) values for PcCel7D were about 1.5 times higher than those for TrCel7A. In case of both TrCel7A and PcCel7D, the k(obs) values for catalytic domains were similar to those for intact enzymes. A model where CBH action is limited by the average length of obstacle-free way on cellulose chain is proposed. Once formed, productive CBH-cellulose complex proceeds with a constant rate determined by the true catalytic constant. After encountering an obstacle CBH will "get stuck" and the rate of further cellulose hydrolysis will be governed by the dissociation rate constant (k(off)), which is low for processive CBH-s.

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The Characterization of the QA Binding Site of the Reaction Center of Rhodopseudomonas sphaeroides

Cited by 23 publications

References 9 publications

In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.

In photosynthetic reaction centers, the free energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity.

Experimental resolution of the free energies of aqueous solvation contributions to ligand-protein binding: quinone-QA site interactions in the photosynthetic reaction center protein.

Mechanism of initial rapid rate retardation in cellobiohydrolase catalyzed cellulose hydrolysis

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