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M R Gunner

Publications and source records attributed to M R Gunner.

At least 19 recordsLinked to original sources
PubMed2005

Modeling binding kinetics at the Q(A) site in bacterial reaction centers.

Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.

Bacterial Proteins↗
PubMed2005

Are acidic and basic groups in buried proteins predicted to be ionized?

Ionizable residues play essential roles in proteins, modulating protein stability, fold and function. Asp, Glu, Arg, and Lys make up about a quarter of the residues in an average protein. Multi-conformation continuum electrostatic (MCCE) calculations were used to predict the ionization states of all acidic and basic residues in 490 proteins. Of all 36,192 ionizable residues, 93.5% were predicted to be ionized. Thirty-five percent have lost 4.08 kcal/mol solvation energy (DeltaDeltaG(rxn)) sufficient to shift a pK(a) by three pH units in the absence of other interactions and 17% have DeltaDeltaG(rxn) sufficient to shift pK(a) by five pH units. Overall 85% of these buried residues (DeltaDeltaG(rxn)>5DeltapK units) are ionized, including 92% of the Arg, 86% of the Asp, 77% of the Glu, and 75% of the Lys. Ion-pair interactions stabilize the ionization of both acids and bases. The backbone dipoles stabilize anions more than cations. The interactions with polar side-chains are also different for acids and bases. Asn and Gln stabilize all charges, Ser and Thr stabilize only acids while Tyr rarely stabilize Lys. Thus, hydroxyls are better hydrogen bond donors than acceptors. Buried ionized residues are more likely to be conserved than those on the surface. There are 3.95 residues buried per 100 residues in an average protein.

Amino Acids, Acidic↗
PubMed2005

Energetics of quinone-dependent electron and proton transfers in Rhodobacter sphaeroides photosynthetic reaction centers.

Proteins bind redox cofactors, modifying their electrochemistry and affinity by specific interactions of the binding site with each cofactor redox state. Photosynthetic reaction centers from Rhodobacter sphaeroides have three ubiquinone-binding sites, Q(A), and proximal and distal Q(B) sites. Ubiquinones, which can be doubly reduced and bind 2 protons, have 9 redox states. However, only Q and Q(-) are seen in the Q(A) site and Q, Q(-), and QH(2) in the proximal Q(B) site. The distal Q(B) function is uncertain. Multiple conformation continuum electrostatics (MCCE) was used to compare the ubiquinone electrochemical midpoints (E(m)) and pK(a) values at these three sites. At pH 7, the Q(A)/Q(A)(-) E(m) is -40 mV and proximal Q(B)/Q(B)(-) -10 mV in agreement with the experimental values (assuming a solution ubiquinone E(m) of -145 mV). Q(B) reduction requires changes in nearby residue protonation and SerL223 reorientation. The distal Q(B)/Q(B)(-) E(m) is a much more unfavorable -260 mV. Q(A) and proximal Q(B) sites generally stabilize species with a -1 charge, while the distal Q(B) site prefers binding neutral species. In each site, the dianion is destabilized because favorable interactions with the residues and backbone increase with charge (q), while the unfavorable loss of solvation energy increases with q(2). Therefore, proton binding before a second reduction, forming QH and then QH(-), is always preferred to forming the dianion (Q(-)(2)). The final product QH(2) is higher in energy at the proximal Q(B) site than in solution; therefore, it binds poorly, favoring release. In contrast, QH(2) binds more tightly than Q at the distal Q(B) site.

Amino Acid Sequence↗
PubMed2003

How cytochromes with different folds control heme redox potentials.

The electrochemical midpoint potentials (E(m)'s) of 13 cytochromes, in globin (c, c(2), c(551), c(553)), four-helix bundle (c', b(562)), alpha beta roll (b(5)), and beta sandwich (f) motifs, with E(m)'s spanning 450 mV were calculated with multiconformation continuum electrostatics (MCCE). MCCE calculates changes in oxidation free energy when a heme-axial ligand complex is moved from water into protein. Calculated and experimental E(m)'s are in good agreement for cytochromes with His-Met and bis-His ligated hemes, where microperoxidases provide reference E(m)'s. In all cytochromes, E(m)'s are raised by 130-260 mV relative to solvated hemes by the loss of reaction field (solvation) energy. However, there is no correlation between E(m) and heme surface exposure. Backbone amide dipoles in loops or helix termini near the axial ligands raise E(m)'s, but amides in helix bundles contribute little. Heme propionates lower E(m)'s. If the propionic acids are partially protonated in the reduced cytochrome, protons are released on heme oxidation, contributing to the pH dependence of the E(m). In all cytochromes studied except b(5)'s and low potential globins, buried side chains raise E(m)'s. MCCE samples ionizable group protonation states, heme redox states, and side chain rotamers simultaneously. Globins show the largest structural changes on heme oxidation and four-helix bundles the least. Given the calculated protein-induced E(m) shift and measured cytochrome E(m) the five-coordinate, His heme in c' is predicted to have a solution E(m) between that of isolated bis-His and His-Met hemes, while the reference E(m) for His-Ntr ligands in cytochrome f should be near that of His-Met hemes.

Bacteria↗
PubMed2003

Calculation of proton transfers in Bacteriorhodopsin bR and M intermediates.

Residue ionization states were calculated in nine crystal structures of bacteriorhodopsin trapped in bR, early M, and late M states by multiconformation continuum electrostatics. This combines continuum electrostatics and molecular mechanics, deriving equilibrium distributions of ionization states and polar residue and water positions. The three central cluster groups [retinal Schiff base (SB), Asp 85 and Asp 212] are ionized in bR structures while a proton has transferred from SB(+) to Asp 85(-) in late M structures matching experimental results. The proton shift in M is due to weaker SB(+)-ionized acid and more favorable SB(0)-ionized acid interactions following retinal isomerization. The proton release cluster (Glu 194 and Glu 204) binds one proton in bR, which is lost to water by pH 8 in late M. In bR the half-ionized state is stabilized by charge-dipole interactions while full ionization is disallowed by charge-charge repulsion between the closely spaced acids. In M the acids move apart, permitting full ionization. Arg 82 movement connects the proton shifts in the central and proton release clusters. Changes in total charge of the two clusters are coupled by direct long-range interactions. Separate calculations consider continuum or explicit water in internal cavities. The explicit waters and nearby polar residues can reorient to stabilize different charge distributions. Proton release to the low-pH, extracellular side of the protein occurs in these calculations where residue ionization remains at equilibrium with the medium. Thus, the key changes distinguishing the intermediates are indeed trapped in the structures.

Aspartic Acid↗
PubMed2002

Exploring the energy landscape for Q(A)(-) to Q(B) electron transfer in bacterial photosynthetic reaction centers: effect of substrate position and tail length on the conformational gating step.

The ability to initiate reactions with a flash of light and to monitor reactions over a wide temperature range allows detailed analysis of reaction mechanisms in photosynthetic reaction centers (RCs) of purple bacteria. In this protein, the electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) is rate-limited by conformational changes rather than electron tunneling. Q(B) movement from a distal to a proximal site has been proposed to be the rate-limiting change. The importance of quinone motion was examined by shortening the Q(B) tail from 50 to 5 carbons. No change in rate was found from 100 to 300 K. The temperature dependence of the rate was also measured in three L209 proline mutants. Under conditions where Q(B) is in the distal site in wild-type RCs, it is trapped in the proximal site in the Tyr L209 mutant [Kuglstatter, A., et al. (2001) Biochemistry 40, 4253-4260]. The electron transfer slows at low temperature for all three mutants as it does in wild-type protein, indicating that conformational changes still limit the reaction rate. Thus, Q(B) movement is unlikely to be the sole, rate-limiting conformational gating step. The temperature dependence of the reaction in the L209 mutants differs somewhat from wild-type RCs. Entropy-enthalpy compensation reduces the difference in rates and free energy changes at room temperature.

Electron Transport↗
PubMed2002

Exploring the energy profile of the Q(A)(-) to Q(B) electron transfer reaction in bacterial photosynthetic reaction centers: pH dependence of the conformational gating step.

Both large- and small-scale conformational changes are needed as proteins carry out reactions. However, little is known about the identity, energy of, and barriers between functional substates on protein reaction coordinates. In isolated bacterial photosynthetic reaction centers, the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), is rate limited by conformational changes at low pH and by proton binding at high pH. The kinetics and thermodynamics of this reaction were determined between 200 and 300 K from pH 6 to pH 10.5. A model with two substates of the reactant, P(+)Q(A)(-)Q(B), one protonated (state A) and one unprotonated (alpha), and one state of the product, P(+)Q(A)Q(B)(-) (B), was able to simulate the dependence of the rate on temperature and pH fairly well. The equilibrium between the three states were measured in situ at each temperature. Proton binding (alpha to A transition) has a favorable DeltaH and unfavorable DeltaS as does the conformational changes required for electron transfer at low pH (A to B). The pK for the A to alpha transition is 9.7 at room temperature, consistent with previous measurements, and equivalent to 13.5 at 200 K. The activation barriers were determined for each transition. Both the alpha to A and the A to B transitions are limited primarily by the activation enthalpy with modest DeltaS.

Electron Transport↗
PubMed2001

Trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria.

In protein, conformational changes are often crucial for function but not easy to observe. Two functionally relevant conformational intermediate states of photosynthetic reaction center protein (RCs) are trapped and characterized at low temperature. RCs frozen in the dark do not allow electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B). In contrast, RCs frozen under illumination in the product (P(+)Q(A)Q(B)(-)) state, with the oxidized electron donor, P(+), and reduced Q(B)(-), return to the ground state at cryogenic temperature in a conformation that allows a high yield of Q(B) reduction. Thus, RCs frozen under illumination are found to be trapped above the ground state in a conformation that allows product formation. When the temperature is raised above 120 K, the protein relaxes to an inactive conformation which is different from the RCs frozen in the dark. The activation energy for this change is 87 +/- 8 meV, and the active and inactive states differ in energy by only 16 +/- 3 meV. Thus, there are several conformational substates along the reaction coordinate with different transition temperatures. The ground state spectra of the RCs in active and inactive conformations report differences in the intraprotein electrostatic field, demonstrating that the dipole or charge distribution has changed. In addition, the electrochromic shift associated with the Q(A)(-) to Q(B) electron transfer at low temperature was characterized. The electron-transfer rate from Q(B)(-) to P(+) was measured at cryogenic temperature and is similar to the rate at room temperature, as expected for an exothermic, electron tunneling reaction in RCs.

Adaptation, Physiological↗
PubMed2000

-deltaG(AB) and pH dependence of the electron transfer from P(+)Q(A)(-)Q(B) toP(+)Q(A)Q(B)(-) in Rhodobacter sphaeroides reaction centers.

The electron transfer from the reduced primary quinone (Q(A)(-)) to the secondary quinone (Q(B)) can occur in two phases with a well-characterized 100 micros component (tau(2)) and a faster process occurring in less than 10 micros (tau(1)). The fast reaction is clearly seen when the native ubiquinone-10 at Q(A) is replaced with naphthoquinones. The dependence of tau(1) on the free-energy difference between the P(+)Q(A)(-)Q(B) and P(+)Q(A)Q(B)(-) states (-) and on the pH was measured using naphthoquinones with different electrochemical midpoint potentials as Q(A) in Rhodobacter sphaeroides reaction centers (RCs) and in RCs where - is changed by mutation of M265 in the Q(A) site from Ile to Thr (M265IT). Q(B) was ubiquinone (UQ(B)) in all cases. Electron transfer was measured by using the absorption differences of the naphthosemiquinone at Q(A) and the ubisemiquinone at Q(B) between 390 and 500 nm. As - was changed from -90 to -250 meV tau(1) decreased from 29 to 0.2 micros. The free-energy dependence of tau(1) provides a reorganization energy of 850 +/- 100 meV for the electron transfer from Q(A)(-) to Q(B). The slower reaction at tau(2) is free-energy independent, so processes other than electron transfer determine the observed rate. The fraction of the reaction at tau(1) increases with increasing driving force and is 100% of the reaction when - is approximately 100 meV more favorable than in the native RCs with ubiquinone as Q(A). The fast phase, tau(1), is pH independent from pH 6 to 11 while tau(2) slows above pH 9. As the Q(A) isoprene tail length is increased from 2 to 10 isoprene units the fraction at tau(1) decreases. However, tau(1), tau(2), and the fraction of the reaction in each phase are independent of the tail length of UQ(B).

Electron Transport↗
PubMed2000

Modeling the effects of mutations on the free energy of the first electron transfer from QA- to QB in photosynthetic reaction centers.

Numerical calculations of the free energy of the first electron transfer in genetically modified reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus were carried out from pH 5 to 11. The multiconformation continuum electrostatics (MCCE) method allows side chain, ligand, and water reorientation to be embedded in the calculations of the Boltzmann distribution of cofactor and amino acid ionization states. The mutation sites whose effects have been modeled are L212 and L213 (the L polypeptide) and two in the M polypeptide, M43(44) and M231(233) in Rb. capsulatus (Rb. sphaeroides). The results of the calculations were compared to the experimental data, and very good agreement was found especially at neutral pH. Each mutation removes or introduces ionizable residues, but the protein maintains a net charge close to that in native RCs through ionization changes in nearby residues. This reduces the effect of mutation and makes the changes in state free energy smaller than would be found in a rigid protein. The state energy of QA-QB and QAQB- states have contributions from interactions among the residues as well as with the quinone which is ionized. For example, removing L213Asp, located in the QB pocket, predominantly changes the free energy of the QA-QB state, where the Asp is ionized in native RCs rather than the QAQB- state, where it is neutral. Side chain, hydroxyl, and water rearrangements due to each of the mutations have also been calculated showing water occupancy changes during the QA- to QB electron transfer.

Alanine↗
PubMed2000

A pragmatic approach to structure based calculation of coupled proton and electron transfer in proteins.

The coupled motion of electrons and protons occurs in many proteins. Using appropriate tools for calculation, the three-dimensional protein structure can show how each protein modulates the observed electron and proton transfer reactions. Some of the assumptions and limitations involved in calculations that rely on continuum electrostatics to calculate the energy of charges in proteins are outlined. Approaches that mix molecular mechanics and continuum electrostatics are described. Three examples of the analysis of reactions in photosynthetic reaction centers are given: comparison of the electrochemistry of hemes in different sites; analysis of the role of the protein in stabilizing the early charge separated state in photosynthesis; and calculation of the proton uptake and protein motion coupled to the electron transfer from the primary (Q(A)) to secondary (Q(B)) quinone. Different mechanisms for stabilizing intra-protein charged cofactors are highlighted in each reaction.

Electrochemistry↗
PubMed2000

Backbone dipoles generate positive potentials in all proteins: origins and implications of the effect.

Asymmetry in packing the peptide amide dipole results in larger positive than negative regions in proteins of all folding motifs. The average side chain potential in 305 proteins is 109 +/- 30 mV (2. 5 +/- 0.7 kcal/mol/e). Because the backbone has zero net charge, the non-zero potential is unexpected. The larger oxygen at the negative and smaller proton at the positive end of the amide dipole yield positive potentials because: 1) at allowed phi and psi angles residues come off the backbone into the positive end of their own amide dipole, avoiding the large oxygen; and 2) amide dipoles with their carbonyl oxygen surface exposed and amine proton buried make the protein interior more positive. Twice as many amides have their oxygens exposed than their amine protons. The distribution of acidic and basic residues shows the importance of the bias toward positive backbone potentials. Thirty percent of the Asp, Glu, Lys, and Arg are buried. Sixty percent of buried residues are acids, only 40% bases. The positive backbone potential stabilizes ionization of 20% of the acids by >3 pH units (-4.1 kcal/mol). Only 6.5% of the bases are equivalently stabilized by negative regions. The backbone stabilizes bound anions such as phosphates and rarely stabilizes bound cations.

Amides↗
PubMed2000

Characterization of a symmetrized mutant RC with 42 residues from the QA site replacing residues in the Q(B) site.

The electron transfer reactions involving Q(A) and Q(B) were investigated in Rb. capsulatus RCs where the Q(B) site was mutated to contain 42 residues from the Q(A) site. The RCs have M220-M261 in the Q(A) site substituted for L193-L227 in the Q(B) site plus the M subunit second-site mutations, M144MI and M145AS, which had been found to restore the ability of the bacteria to grow photosynthetically. These mutants lack L210D, L212E, L213D, and L223S which have been previously shown to affect the electron transfer from Q(A) (-) to Q(B). Despite the large change in the Q(B) pocket, secondary quinone function still can be reconstituted. The UQ(4) dissociation constant for the Q(B) site in the mutant is only three times as large as in the wild type RCs. The rate of charge recombination (P(+)Q(A)Q(B) (-) --> PQ(A)Q(B)) (k (BP)) is reduced from 8.9 s(-1) in wild type RCs to 0.05s(-1) in the mutant, This indicates that Q(A)Q(B) (-) is stabilized relative to Q(A) (-)Q(B) by at least 60 meV more than in wild type protein. k (BP) is pH independent in the mutant RCs, while in wild type RCs k (BP) increases at alkaline pHs as reduction of Q(B) becomes energetically less favorable. Similar pH independent, slow k (BP) has been found in the L212EA/L213DA double mutant. The largest change found in the mutant is that the electron transfer from Q(A) (-) to Q(B) (k (AB) ((1)) approximately 14 s(-1)) is 3 orders of magnitude slower than in wild type RCs (10(4) s(-1)).

Journal Article↗
PubMed1999

Calculated protein and proton motions coupled to electron transfer: electron transfer from QA- to QB in bacterial photosynthetic reaction centers.

Reaction centers from Rhodobacter sphaeroides were subjected to Monte Carlo sampling to determine the Boltzmann distribution of side-chain ionization states and positions and buried water orientation and site occupancy. Changing the oxidation states of the bacteriochlorophyll dimer electron donor (P) and primary (QA) and secondary (QB) quinone electron acceptors allows preparation of the ground (all neutral), P+QA-, P+QB-, P0QA-, and P0QB- states. The calculated proton binding going from ground to other oxidation states and the free energy of electron transfer from QA-QB to form QAQB- (DeltaGAB) compare well with experiment from pH 5 to pH 11. At pH 7 DeltaGAB is measured as -65 meV and calculated to be -80 meV. With fixed protein positions as in standard electrostatic calculations, DeltaGAB is +170 meV. At pH 7 approximately 0.2 H+/protein is bound on QA reduction. On electron transfer to QB there is little additional proton uptake, but shifts in side chain protonation and position occur throughout the protein. Waters in channels leading from QB to the surface change site occupancy and orientation. A cluster of acids (GluL212, AspL210, and L213) and SerL223 near QB play important roles. A simplified view shows this cluster with a single negative charge (on AspL213 with a hydrogen bond to SerL233) in the ground state. In the QB- state the cluster still has one negative charge, now on the more distant AspL210. AspL213 and SerL223 move so SerL223 can hydrogen bond to QB-. These rearrangements plus other changes throughout the protein make the reaction energetically favorable.

Benzoquinones↗
PubMed1998

Kinetic phases in the electron transfer from P+QA-QB to P+QAQB- and the associated processes in Rhodobacter sphaeroides R-26 reaction centers.

Electron transfer from P+QA-QB to form P+QAQB- was measured in Rhodobacter sphaeroides R-26 reaction centers (RCs) where the native primary quinone, ubiquinone-10 (UQA), was replaced by 2-methyl-3-phytyl-1,4-naphthoquinone (MQA). The native secondary quinone, UQ-10, was retained as UQB. The difference spectrum of the semiquinone MQA- minus UQB- absorption is very similar to that of MQ- minus UQ- in solution (398-480 nm). Thus, the absorption change provides a direct monitor of the electron transfer from MQA- to UQB. In contrast, when both QA and QB are UQ-10 the spectral difference between UQA- and UQB- arises from electrochromic responses of RC chromophores. Three kinetic processes are seen in the near UV (390-480 nm) and near-IR (740-820 nm). Analysis of the time-correlated spectra support the conclusion that the changes at tau1 approximately 3 micros are mostly due to electron transfer, electron transfer and charge compensation are mixed in tau2 approximately 80 micros, while little or no electron transfer occurs at 200-600 micros (tau3) in MQAUQB RCs. The 80-micros rate has been previously observed, while the fast component has not. The fast phase represents 60% of the electron-transfer reaction (398 nm). The activation energy for electron transfer is DeltaG approximately 3.5 kcal/mol for both tau1 and tau2 between 0 and 30 degrees C. In isolated RCs with UQA, if there is any fast component, it appears to be faster and less important than in the MQA reconstituted RCs.

Electron Transport↗
PubMed1997

The contribution of electrostatic and van der Waals interactions to the stereospecificity of the reaction catalyzed by lactate dehydrogenase.

Continuum electrostatic calculations in conjunction with molecular dynamics simulations have been used to investigate the source of the stereospecificity in the hydride transfer reaction catalyzed by lactate dehydrogenase (LDH). These studies show that favorable electrostatic interactions between the carboxamide group of the reduced nicotinamide adenine dinucleotide coenzyme and protein residues of the active site of LDH can account for much if not all of the stereospecificity of the LDH-catalyzed reaction, with A-side hydride transfer more than 10(7) times greater than B-side transfer. Unfavorable steric interactions within the binding complex for B-side transfer are not found.

Animals↗
PubMed1997

Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties.

A method for combining calculations of residue pKa's with changes in the position of polar hydrogens has been developed. The Boltzmann distributions of proton positions in hydroxyls and neutral titratable residues are found in the same Monte Carlo sampling procedure that determines the amino acid ionization states at each pH. Electrostatic, Lennard-Jones potentials, and torsion angle energies are considered at each proton position. Many acidic and basic residues are found to have significant electrostatic interactions with either a water- or hydroxyl-containing side chain. Protonation state changes are coupled to reorientation of the neighboring hydroxyl dipoles, resulting in smaller free energy differences between neutral and ionized residues than when the protein is held rigid. Multiconformation pH titration gives better agreement with the experimental pKa's for triclinic hen egg lysozyme than conventional rigid protein calculations. The hydroxyl motion significantly increases the protein dielectric response, making it sensitive to the composition of the local protein structure. More than one conformer per residue is often found at a given pH, providing information about the distribution of low-energy lysozyme structures.

Entropy↗