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Biomedical subjects

P L Dutton

Publications and source records attributed to P L Dutton.

At least 55 records · Page 3Linked to original sources
PubMed1995

Tyrosine 147 of cytochrome b is required for efficient electron transfer at the ubihydroquinone oxidase site (Qo) of the cytochrome bc1 complex.

In Rhodobacter capsulatus, tyrosine (Y) 147 is a highly conserved residue of the cyt b subunit of the bc1 complex. It is located in the vicinity of residues altered in spontaneous inhibitor resistant mutants that affect the ubihydroquinone oxidase (Qo) site of this enzyme. In this work, Y147 was substituted with phenylalanine (F), valine (V), serine (S), and alanine (A) using site-directed mutagenesis in an effort to investigate its specific role in the Qo site. Of the four mutants obtained, Y147S and Y147A exhibited very low ubihydroquinone:cyt c reductase activities and were unable to support photosynthetic growth (Ps) while Y147F and Y147V were Ps+. In all mutants, no changes in the redox midpoint potentials (Em7) of the cyt bH and cyt bL, the occupancy of the Qo site by Q/QH2, and the flash-induced reverse electron transfer kinetics from Qi to cyt bH were observed. On the other hand, rates of electron transfer from Qo to cyt bH were mildly reduced (2-3-fold) in Y147F and V but dramatically decreased (about 20-fold) in Y147A and S, localizing the defect to the Qo site. Thus, Y147A and S are members of a novel class of Qo site mutants that affect the Qo site catalysis without perturbing the accessibility or binding of the substrate. Additional insight to the role of Y147 on ubihydroquinone oxidation was gained by analyzing the Ps+ revertants of these mutants. Two pseudorevertants contained a second mutation [isoleucine (I) or valine (V)] at the highly conserved M154 position, six residues away from Y147.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Arabidopsis↗
PubMed1995

Biological electron transfer.

Many oxidoreductases are constructed from (a) local sites of strongly coupled substrate-redox cofactor partners participating in exchange of electron pairs, (b) electron pair/single electron transducing redox centers, and (c) nonadiabatic, long-distance, single-electron tunneling between weakly coupled redox centers. The latter is the subject of an expanding experimental program that seeks to manipulate, test, and apply the parameters of theory. New results from the photosynthetic reaction center protein confirm that the electronic-tunneling medium appears relatively homogeneous, with any variances evident having no impact on function, and that control of intraprotein rates and directional specificity rests on a combination of distance, free energy, and reorganization energy. Interprotein electron transfer between cytochrome c and the reaction center and in lactate dehydrogenase, a typical oxidoreductase from yeast, are examined. Rates of interprotein electron transfer appear to follow intraprotein guidelines with the added essential provision of binding forces to bring the cofactors of the reacting proteins into proximity.

Electron Transport↗
PubMed1995

Binding of halothane to serum albumin demonstrated using tryptophan fluorescence.

BACKGROUND: The site of action of general anesthesia remains controversial, but evidence in favor of specific protein target(s) is accumulating. Saturable binding of halothane to bovine serum albumin (BSA) has recently been reported using photoaffinity labeling and fluorine 19 nuclear magnetic resonance spectroscopy. We report a new approach to study anesthetic binding to soluble proteins, based on native tryptophan fluorescence. METHODS: Thymol-free halothane and fatty acid-free BSA were equilibrated in gas-tight Hamilton syringes and dispensed into stoppered quartz cuvettes at predetermined dilutions. Steady-state fluorescence spectroscopy was used to study their interaction. RESULTS: Halothane quenched the tryptophan fluorescence of BSA in a concentration-dependent, saturable manner with a dissociation constant = 1.8 +/- 0.2 mM and a Hill number = 1.0 +/- 0.1. The two optical isomers of halothane bound to BSA with equal affinity. The ability of halothane to quench BSA tryptophan fluorescence was markedly decreased at pH 3.0 (which causes full uncoiling of BSA), with loss of saturable binding. Diethyl ether displaced a portion of halothane from its binding sites. Circular dichroism spectroscopy revealed no significant effect of halothane or diethyl ether on the secondary structure of BSA. CONCLUSIONS: The results suggest that halothane binds in hydrophobic domains containing tryptophan in BSA. This approach may prove useful for studying the interaction of volatile anesthetics and proteins and has the advantage that the location of halothane in the protein is identified.

Fluorescence↗
PubMed1994

Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.

Protein and cofactor vibrational dynamics associated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femto-second (300-400 fs) time-resolved infrared (1560-1960 cm-1) spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation modifies the polypeptide carbonyl spectra. The potential energy surfaces of the "special pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process. Kinetic signatures of all other transient intermediates, P, P*, and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.

Kinetics↗
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↗
PubMed1994

Design and synthesis of multi-haem proteins.

A water-soluble, 62-residue, di-alpha-helical peptide has been synthesized which accommodates two bis-histidyl haem groups. The peptide assembles into a four-helix dimer with 2-fold symmetry and four parallel haems that closely resemble native haems in their spectral and electrochemical properties, including haem-haem redox interaction. This protein is an essential intermediate in the synthesis of molecular 'maquettes', a novel class of simplified versions of the metalloproteins involved in redox catalysis and in energy conversion in respiratory and photosynthetic electron transfer.

Amino Acid Sequence↗
PubMed1994

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

Amino Acid Sequence↗
PubMed1993

Picosecond infrared studies of the dynamics of the photosynthetic reaction center.

The changes in the vibrational transitions of the protein and redox cofactors of the photosynthetic reaction center were examined by picosecond infrared spectroscopy. The spectra in the vibrational mid-infrared region (1800-1550 cm-1) of hydrated and partially dehydrated reaction centers were investigated from 50 ps to 4 ns after photoinitiation of the electron transfer. Features in the infrared difference spectra were identified with both protein and redox cofactor vibrational modes and correlated with electron transfer events whose kinetics were measured in the infrared and visible regions. The observed protein response is confined to a few amide I transitions (1644 cm-1, 1661 cm-1, 1665 cm-1) and carboxylic residues (1727 cm-1). About 85% of the observed signal corresponded to alterations in the cofactor-associated ester and keto carbonyls. The amide I and carboxylic transitions appeared prior to 50 ps, suggesting that the primary electron transfer event is coupled with a specific piece of the protein backbone and to glutamic or aspartic residues nearby the special pair. Infrared absorption changes accompanying bacteriochlorophyll-dimer cation formation dominated the signal at all times investigated. Infrared spectral changes observed in hydrated and partially dehydrated reaction centers were distinctly different; a band at 1665 cm-1 with a spectral width of 6 cm-1 in the hydrated protein, corresponding to a protein amide I bleach, was not present in the dehydrated film. These differences are discussed in terms of the markedly different electron transfer kinetics observed in the presence of water.

Electron Transport↗
PubMed1993

Influence of QA site redox cofactor structure on equilibrium binding, in situ electrochemistry, and electron-transfer performance in the photosynthetic reaction center protein.

The native ubiquinone-10 redox cofactor has been removed from the QA site of the isolated reaction center protein from Rhodobacter sphaeroides and reconstitution attempted with 28 non-quinone molecules in order to identify factors governing cofactor function and the selectivity displayed by the site in the electron transfers that it catalyzes. Equilibrium binding, in situ electrochemistry, and the kinetics of electron transfer to and from the QA site occupant were examined. Four classes of non-quinone molecules are distinguished according to their ability to occupy the QA site and conduct intraprotein electron transfers. The minimal requirements for occupancy of the QA site are at least one ring and a heteroatom hydrogen bond acceptor. Thus, binding at the site is not highly selective. The rates of electron transfers to and from the class of non-quinone molecules (four) that satisfy the criteria for cofactor function at the QA site compare well with rates previously determined from 14 to 295 K for 14 quinone replacements with comparable values of the reaction free energy. This indicates that the rates are relatively insensitive to variations in exotic and quinone cofactor reorganization energy and the vibrational frequencies coupled to the electron transfers, and that the exotic and quinone cofactors are bound in the QA site in comparable positions. It appears that any variation in rate is determined predominantly by the value of the reaction free energy. The QA site protein-cofactor solvation contribution to the in situ electrochemical potential is roughly constant for 12 rigid quinone and 2 exotic cofactors (average value-61 +/- 2 kcal/mol). Favorable electrostatic contributions governing the reaction free energy are therefore also relatively insensitive to cofactor structure. However, flexible molecules appear to encounter in situ steric constraints that lower the electron affinity by destabilizing the reduced cofactor species. This is a strong determinant of whether a molecule, once in the QA site, will function. These findings compare well with those from studies of electron transfers in synthetic systems.

Bacteriochlorophylls↗
PubMed1993

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

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 (delta G degrees B,w), in hexane solution containing 30 mM water (delta G degrees B,hh), and after partial dehydration (delta G degrees B,dh) with magnesium sulfate. Our aim is to resolve the contributions of aqueous bulk phase solvation and protein hydration contributions to binding in order to characterize in detail the direct interactions between the ligands and protein at the QA site. This is accomplished by comparing the differences between delta G degrees B,w and delta G degrees B,hh (or delta G degrees B,dh) with the water to hexane solvent transfer free energies of the quinones (delta G degrees tr,Q). Values of delta G degrees tr,Q are determined separately in binary solution and range from 0.65 to -5.69 kcal/mol (1 cal = 4.184 J). The results are interpreted in terms of a thermodynamic cycle that links the species involved in the binding and solvent transfer equilibria. Values of delta G degrees B,hh -delta G degrees B,w are linearly correlated with -delta G degrees tr,Q (slope, 0.78 +/- 0.04; ordinate intercept, -0.13 +/- 0.12 kcal/mol). The deviation of the experimental slopes from the predicted value of unity is attributed in part to a systematic decrease of quinone thermodynamic activity in the aqueous binding medium relative to the aqueous phase in the binary partitioning solvent system. The difference between the quinone-QA site binding free energies measured in hydrated hexane and water is therefore related only to the difference in bulk phase quinone solvation, as given by 0.78 delta G degrees tr,Q. The linear relation obtained using delta G degrees B,dh -delta G degrees B,w has the same slope, but the intercept is decreased to -1.48 +/- 0.19 kcal/mol, indicating that quinone binding strengths in the hexane system are uniformly enhanced after partial dehydration. This suggests that the quinones encounter a common opposition to interaction with the site in the hydrated, relative to the partially dehydrated, state. The further utility of the method to directly assess ligand-site binding free energies is demonstrated with examples that address the contributions of molecular size and dipolar or hydrogen bond interactions to the binding of quinones at the QA site.

Hexanes↗
PubMed1993

Hydroubiquinone-cytochrome c2 oxidoreductase from Rhodobacter capsulatus: definition of a minimal, functional isolated preparation.

The hydroubiquinone-cytochrome c2 oxidoreductase (cyt bc1) from Rhodobacter capsulatus has been solubilized according to the dodecyl maltoside method and isolated, and its minimal functional composition has been characterized. We find the complex to be composed of three protein subunits corresponding to polypeptides of cyt b (44 kDa), cyt c1 (33 kDa), and 2Fe2S cluster (24 kDa). A fourth band sometimes discernable at 22 kDa appears to be an artifact of the polyacrylamide gel electrophoresis procedure. Its appearance is shown to be derived from the 2Fe2S cluster subunit by the similarity of the binding of subunit-specific monoclonal antibodies and the identical N-terminal sequence of the 24- and 22-kDa bands. The cofactors of cyt bc1, namely, cyt bH, cyt bL, cyt c1, and the 2Fe2S center, the Qos and Qow domains of the Qo site, and the Qi site appear intact as indicated by their optical and EPR spectral signatures, redox properties, and inhibitor binding. The electron paramagnetic resonance spectrum of the cyt bH heme is altered by antimycin, consistent with a change in the dihedral angle between the ligating histidine imidazoles, while the spectrum of the cyt bL heme is broadened by stigmatellin. The ubiquinone-10 content is variable, ranging from 0.8 to 3 molecules/cyt bc1. Activity studies define this three-subunit cyt bc1 complex as a minimal structure, equipped as the enzyme in the native state and capable of full catalytic activity.

Amino Acid Sequence↗
PubMed1992

Engineering protein structure for electron transfer function in photosynthetic reaction centers.

A basic relationship is defined that incorporates the three parameters that effectively modulate the rate of intraprotein electron transfer, namely distance, free energy and reorganization energy. This empirically validated relationship is used to explore the minimal requirements for protein-catalyzed conversion of excited electronic states into stable charge separated states, the essence of photosynthesis.

Electron Transport↗
PubMed1992

Cytochrome bc1 complex [2Fe-2S] cluster and its interaction with ubiquinone and ubihydroquinone at the Qo site: a double-occupancy Qo site model.

The ubiquinone complement of Rhodobacter capsulatus chromatophore membranes has been characterized by its isooctane solvent extractability and electrochemistry; we find that the main ubiquinone pool (Qpool) amounts to about 80% of the total ubiquinone and has an Em7 value close to 90 mV. To investigate the interactions of ubiquinone with the cyt bc1 complex, we have examined the distinctive EPR line shapes of the [2Fe-2S] cluster of the cyt bc1 complex when the Qpool-cyt bc1 complex interactions are modulated by changing the numbers of Q or QH2 present (by solvent extraction and reconstitution), by the exposure of the [2Fe-2S] to the Qpool in different redox states, by the presence of inhibitors specific for the Qo site (myxothiazol and stigmatellin) and Qi site (antimycin), and by site-specific mutations of side chains of the cyt b polypeptide (mutants F144L and F144G) previously identified as important for Qo site structure. Evidence suggests that the Qo site can accommodate two ubiquinone molecules. One (designated Qos) is bound relatively strongly and is second only to the ubiquinone of the QA site of the reaction center in its resistance to solvent extraction. In this strong interaction, the Qo site binds Q and QH2 with approximately equal affinities. Their bound states are distinguished by their effects on the [2Fe-2S] cluster spectral feature at gx at 1.783 (Q) and gx at 1.777 (QH2); titration of the line-shape change reveals an Em7 value of approximately 95 mV. The other molecule (Qow) is bound more weakly, in the same range as the ubiquinone of the QB site of the reaction center. Again, the affinities of the Q form (gx at 1.800) and QH2 form (gx at 1.777) are nearly equal, and the Em7 value measured is approximately 80 mV. These results are discussed in terms of earlier EPR analyses of the cyt bc1 complexes of other systems. A Qo site double-occupancy model is considered that builds on the previous model based on Qo site mutants [Robertson, D. E., Daldal, F.,& Dutton, P. L. (1990) Biochemistry 29, 11249-11260] and includes the recent suggestion that two of the [2F3-2S] cluster ligands of the R. capsulatus cyt bc1 complex are histidines [Gurbiel, R. J. Ohnishi, T., Robertson, D. E. Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. We speculate that the cyt bc1 complex complexes a full enzymatic turnover without necessary exchange of ubiquinone with the Qpool.

Bacterial Chromatophores↗
PubMed1992

Nature of biological electron transfer.

Powerful first-order analysis of intraprotein electron transfer is developed from electron-transfer measurements both in biological and in chemical systems. A variation of 20 A in the distance between donors and acceptors in protein changes the electron-transfer rate by 10(12)-fold. Protein presents a uniform electronic barrier to electron tunnelling and a uniform nuclear characteristic frequency, properties similar to an organic glass. Selection of distance, free energy and reorganization energy are sufficient to define rate and directional specificity of biological electron transfer, meeting physiological requirements in diverse systems.

Animals↗
PubMed1991

Langmuir-Blodgett monolayer films of bacterial photosynthetic membranes and isolated reaction centers: preparation, spectrophotometric and electrochemical characterization.

The Langmuir-Blodgett (LB) film technique has been successfully applied to the construction of stable and photo-active films of chromatophore membranes and isolated reaction centers from two species of photosynthetic bacteria, Rhodobacter sphaeroides and Rhodopseudomonas viridis. LB films of these preparations were characterized at the air/water interface through compression isotherms and film stabilities. Films deposited on glass slides were analyzed by spectrophotometric and redox potentiometric techniques. The results obtained indicate that the in vivo properties of the photosynthetic apparatus in the deposited films are essentially unchanged. Furthermore, the pigments and redox cofactors in the films are highly oriented and offer a unique opportunity for structural and functional studies of the kind described in the accompanying paper (Biochim. Biophys. Acta 1057 (1991) 258-272).

Cytochrome c Group↗