Biomedical subjects

M R Gunner

Publications and source records attributed to M R Gunner.

29 records · Page 2Linked to original sources

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↗

PubMed1996

Calculated coupling of electron and proton transfer in the photosynthetic reaction center of Rhodopseudomonas viridis.

Based on new Rhodopseudomonas (Rp.) viridis reaction center (RC) coordinates with a reliable structure of the secondary acceptor quinone (QB) site, a continuum dielectric model and finite difference technique have been used to identify clusters of electrostatically interacting ionizable residues. Twenty-three residues within a distance of 25 A from QB (QB cluster) have been shown to be strongly electrostatically coupled to QB, either directly or indirectly. An analogous cluster of 24 residues is found to interact with QA (QA cluster). Both clusters extend to the cytoplasmic surface in at least two directions. However, the QB cluster differs from the QA cluster in that it has a surplus of acidic residues, more strong electrostatic interactions, is less solvated, and experiences a strong positive electrostatic field arising from the polypeptide backbone. Consequently, upon reduction of QA or QB, it is the QB cluster, and not the QA cluster, which is responsible for substoichiometric proton uptake at neutral pH. The bulk of the changes in the QB cluster are calculated to be due to the protonation of a tightly coupled cluster of the three Glu residues (L212, H177, and M234) within the QB cluster. If the lifetime of the doubly reduced state QB2- is long enough, Asp M43 and Ser L223 are predicted to also become protonated. The calculated complex titration behavior of the strongly interacting residues of the QB cluster and the resulting electrostatic response to electron transfer may be a common feature in proton-transferring membrane protein complexes.

Amino Acid Sequence↗

PubMed1995

Volume contraction on photoexcitation of the reaction center from Rhodobacter sphaeroides R-26: internal probe of dielectrics.

Reaction centers of Rhodobacter sphaeroides undergo a approximately 20 A3/mole volume contraction in < 50 ns after excitation. The rapid volume change is tentatively assigned to electrostriction. From its magnitude, we infer that the effective dielectric coefficient is 10-15 if the compressibility of the reaction center is similar to that of globular proteins. The volume contraction is not sensitive to replacement of the natural ubiquinone at the QA site by other quinones or to the occupancy of the QB site. The quenching caused by pressure on the reaction centers most likely occurs on a faster time scale than that of electron transfer.

Biophysical Phenomena↗

PubMed1994

Influence of hydrocarbon tail structure on quinone binding and electron-transfer performance at the QA and QB sites of the photosynthetic reaction center protein.

Binding free energies of 37 functional replacement quinone cofactors with systematically altered hydrocarbon tail structures have been determined for the QA and QB redox catalytic sites of the reaction center protein isolated from Rhodobacter sphaeroides and solubilized in aqueous and in hexane solutions. The first two and part of the third isoprene units of the 10-unit tail of the native ubiquinone-10 cofactor interact with the protein interior at each site. Contributions of the same tail structures to the binding free energies of quinones at the QA and QB sites are comparable, suggesting that the binding domains share common features. Comparison of the affinities of a homologous series of 10 n-alkyl-substituted ubiquinones resolves the binding forces along the length of the tail binding domain and shows that strong steric constraints oppose accommodation of the tail in its extended conformation. Differences in the contributions of identical tail substituents to ubiquinone- and menaquinone-QA site affinities, and tail-induced changes of up to 5-fold in the rates of QA site-mediated electron-transfer reactions, suggest that the tail adjusts the position of the quinone ring. Substitution of ubiquinone with the native 10-unit isoprene tail does not alter the affinity for the sites as determined in hexane solution. However, one- and two-isoprene-substituted quinones bind more tightly than analogs substituted with saturated-alkyl tail substituents. The sites therefore exhibit binding specificity for the native isoprene tail structure. Calculations indicate that the binding specificity arises primarily from a lower integrated torsion potential energy in the bound isoprene tails. The results suggest that the in vivo tail-protein interaction is designed to deter competitive interference of quinone function by amphiphilic species present in the native membrane.

Crystallography, X-Ray↗

PubMed1993

Electron-transfer kinetics and electrostatic properties of the Rhodobacter sphaeroides reaction center and soluble c-cytochromes.

The kinetics of electron transfer between the Rhodobacter sphaeroides R-26 reaction center and nine soluble c-cytochromes have been analyzed and compared to the patterns of the surface electrostatic potentials for each of the proteins. Characteristic first-order electron-transfer rates for 1:1 complexes formed at low ionic strength between the reaction center and the different c-cytochromes were identified and found to vary by a factor of almost 100, while second-order rates were found to differ by greater than 10(6). A correlation was found between the location of likely electrostatic interaction domains on each cytochrome and its characteristic rate of electron transfer. The interaction domains were identified by mapping electrostatic potentials, calculated from the Poisson-Boltzmann equation, onto simulated "encounter surfaces" for each of the cytochromes and the reaction center. For the reaction center, the c-cytochrome binding domain was found to have almost exclusively net negative potential (< -3 kT) and to be shifted slightly toward the M-subunit side of the reaction center. The location of interaction domains of complementary, positive potential (> 3 kT) differed for each cytochrome. The correspondence between electrostatic, structural, and kinetic properties of 1:1 reaction center-cytochrome complexes leads to a proposed mechanism for formation of reaction center-cytochrome electron-transfer complexes that is primarily driven by the juxtaposition of regions of delocalized complementary potential. In this mechanism the clustering of charged residues is of primary importance and not the location of specific residues. A consequence of this mechanism is that many different sets of charge distributions are predicted to be capable of stabilizing a specific configuration for a reaction center-cytochrome complex. This mechanism for reaction center association with water-soluble c-cytochromes fits molecular recognition mechanisms proposed for c-cytochromes in nonphotosynthetic systems. In general, the kinetic scheme for reaction center driven cytochrome oxidation was found to vary between a simple two-state model, involving cytochrome in free and reaction center bound states, and a three-state model, that includes cytochrome binding in kinetically competent ("proximal") and incompetent ("distal") modes. The kinetically incompetent mode of cytochrome binding is suggested not to be an intrinsic feature of the reaction center-cytochrome association but is likely to be due to variation in the physical state of the reaction center.

Animals↗

PubMed1993

On the calculation of pKas in proteins.

This paper describes a general method to calculate the pKas of ionizable groups in proteins. Electrostatic calculations are carried out using the finite difference Poisson-Boltzmann (FDPB) method. A formal treatment of the calculation of pKas within the framework of the FDPB method is presented. The major change with respect to previous work is the specific incorporation of the complete charge distribution of both the neutral and charged forms of each ionizable group into the formalism. This is extremely important for the treatment of salt bridges. A hybrid statistical mechanical/Tanford-Roxby method, which is found to be significantly faster than previous treatments, is also introduced. This simplifies the problem of summing over the large number of possible ionization states for a complex polyion. Applications to BPTI and serine proteases suggest that the calculations can be quite reliable. However, the necessity of including bound waters in the treatment of the Asp-70... His-31 salt bridge in T4 lysozyme and experience with other proteins suggest that additional factors ultimately need to be considered in a comprehensive treatment of pKas in proteins.

Aprotinin↗

PubMed1991

Electrostatic control of midpoint potentials in the cytochrome subunit of the Rhodopseudomonas viridis reaction center.

The photosynthetic reaction center of Rhodopseudomonas viridis has four hemes in a linear arrangement with alternating high- and low-potential sites. Their midpoints are -60, 20, 310, and 380 mV [Dracheva, S. M., Drachev, L. A., Konstantinov, A. A., Semenov, A. Y., Skulachev, V. P., Arutjunjan, A. M., Shuvalov, V. A. & Zaberezhnaya, S. M. (1988) Eur. J. Biochem. 171, 253-264]. Electrostatic calculations reproduce the 440-mV midpoint spread and assignments of high- and low-potential hemes. When calculations on model compounds to connect the theoretical midpoints to the standard hydrogen electrode are used, the absolute electrochemical midpoints for the reaction center hemes are also in good agreement with experiment. The free energy of oxidation is found to be dependent on pairwise interactions with charged amino acids, heme propionic acids, previously oxidized hemes, and axial ligands and on the reaction field induced by heme oxidation.

Amino Acid Sequence↗

PubMed1986

Radical-pair energetics and decay mechanisms in reaction centers containing anthraquinones, naphthoquinones or benzoquinones in place of ubiquinone.

In reaction centers from Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides), light causes an electron-transfer reaction that forms the radical pair state (P+I-, or PF) from the initial excited singlet state (P) of a bacteriochlorophyll dimer (P). Subsequent electron transfer to a quinone (Q) produces the state P+Q-. Back electron transfer can regenerate P from P+Q-, giving rise to 'delayed' fluorescence that decays with approximately the same lifetime as P+Q-. The free-energy difference between P+Q- and P can be determined from the initial amplitude of the delayed fluorescence. In the present work, we extracted the native quinone (ubiquinone) from Rps. sphaeroides reaction centers, and replaced it by various anthraquinones, naphthoquinones, and benzoquinones. We found a rough correlation between the halfwave reduction potential (E1/2) of the quinone used for reconstitution (as measured polarographically in dimethylformamide) and the apparent free energy of the state P+Q- relatively to P. As the E1/2 of the quinone becomes more negative, the standard free-energy gap between P+Q- and P decreases. However, the correlation is quantitatively weak. Apparently, the effective midpoint potentials (Em) of the quinones in situ depend subtly on interactions with the protein environment in the reaction center. Using the value of the Em for ubiquinone determined in native reaction centers as a reference, and the standard free energies determined for P+Q- in reaction centers reconstituted with other quinones, the effective Em values of 12 different quinones in situ are estimated. In native reaction centers, or in reaction centers reconstituted with quinones that give a standard free-energy gap of more than about 0.8 eV between P+Q- and P*, charge recombination from P+Q- to the ground state (PQ) occurs almost exclusively by a temperature-insensitive mechanism, presumably electron tunneling. When reaction centers are reconstituted with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 eV, part or all of the decay proceeds through a thermally accessible intermediate. There is a linear relationship between the log of the rate constant for the decay of P+Q- via the intermediate state and the standard free energy of P+Q-. The higher the free energy, the faster the decay. The kinetic and thermodynamic properties of the intermediate appear not to depend strongly on the quinone used for reconstitution, indicating that the intermediate is probably not simply an activated form of P+Q-.(ABSTRACT TRUNCATED AT 400 WORDS)

Anthraquinones↗

PubMedDate not supplied

pH dependence of heme electrochemistry in cytochromes investigated by multiconformation continuum electrostatic calculations.

Cytochromes belong to a diverse family of heme-containing redox proteins that function as intermediaries in electron transfer chains. They can be soluble, extrinsic, or intrinsic membrane proteins, and are found in different structural motifs (globin, 4-helix bundles, alpha beta roll, beta sandwich). Measured electrochemical midpoint potentials vary over a wide range even though the basic redox reaction at the heme is the same for all cytochromes. The perturbation of the heme electrochemistry is induced by the protein structure. Also, the pH dependence varies since it depends on the strength of interaction between the heme and surrounding residues as well as the ionization states of these groups. Multiconformation continuum electrostatics (MCCE) has been used to investigate the pH dependence of heme electrochemistry in cytochromes with different folds. Often propionates are the primary contributors for pH dependence especially if they are partially protonated in the reduced heme as it is shown for globin cytochrome c551 P. aeruginosa and cytochrome b5 R. norvegicus (alpha beta roll). However, if the propionates are already fully ionized at a certain pH they do not contribute to the pH dependence even if they have big interaction with the heme. At pH 7 there is no propionate contribution for cytochrome f C. reinhardtii (beta sandwich) and the 4-helix bundle c' R. palustris. Other residues can also change their ionization significantly during heme oxidation and therefore be involved in proton release and pH dependence. These residues have been identified for different cytochrome types.

Amino Acid Motifs↗