PHD: predicting one-dimensional protein structure by profile-based neural networks.
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Amino Acid Sequence↗
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Effects of chaotropic anions on the distribution of conformational substates of amicyanin, wild type and Cys3Ala/Cys26Ala azurin mutant.
The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric <--> g and <--> A tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins. Such a reduction, quantified by the standard deviations sigma(g axially) and sigma(A axially) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(g axially) and sigma(A axially) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N(-)(3) and SCN(-) addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions.
Amino Acid Substitution↗
Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions.
Sulfur-utilizing denitrification can be performed by denitrifying sulfur bacteria under autotrophic and heterotrophic conditions. To investigate the effect of organics (methanol and landfill leachate) on sulfur-utilizing denitrification, six laboratory-scale sulfur packed columns were operated under autotrophic, mixotrophic and heterotrophic conditions for approximately 1 year. The performance of the columns was monitored by measuring the pH, nitrate, nitrite, sulfate, sulfide, alkalinity dissolved organic carbon (DOC), and turbidity. These tests indicated that the mixotrophic column had a higher nitrate removal capacity than the autotrophic column. It was also found that under mixotrophic conditions, some portion of nitrate was removed heterotrophically and the remainder was denitrified by sulfur-utilizing autotrophic bacteria without inhibition by organics. In addition, sulfate production and alkalinity consumption were reduced under mixotrophic conditions. These results suggest that mixotrophic conditions that allow simultaneous reactions of autotrophic and heterotrophic denitrification provide significant advantages in terms of nitrate and DOC removal, sulfate production decrease, and alkalinity consumption over obligate autotrophic or heterotrophic denitrification.
Bacteria↗
Reduction of perchlorate and nitrate by salt tolerant bacteria.
Spent regenerant brine from ion-exchange technology for the removal of perchlorate and nitrate produces a high salt waste stream, which requires remediation before disposal. Bioremediation is an attractive treatment option. In this study, we enriched for salt tolerant bacteria from sediments from Cargill salt evaporation facility (California, USA), the Salton Sea (California, USA), and a high density hydrocarbon oxidizing bacterial cocktail. The bacterial cocktail enrichment culture reduced ClO4- from 500 to 260 mg 1 in 4 weeks. Salt tolerant bacterial isolates from the enrichment cultures and two denitrifying salt tolerant bacteria, Haloferax denitrificans and Parococcus halodenitricans, substantially reduced perchlorate. The highest rate of perchlorate removal was recorded with the isolate, Citrobacter sp.: 32% reduction in 1 week. This bacterium substantially reduced perchlorate in 0-5% NaCl solutions and maximally at 30 degrees C and at an initial pH 7.5. In simulated brines containing 7.5% total solids, the Citrobacter sp. significantly reduced both perchlorate and nitrate with 34.9 and 15.6% reduction, respectively, in 1 week. Coculture of a potent perchlorate reducing, non-salt tolerant (non-saline) bacterium, perclace and the Citrobacter sp. proved most effective for perchlorate removal in the brine (46.4% in 1 week). This study demonstrates that both anions can be reduced in treatment of brines from ion exchange systems.
Biodegradation, Environmental↗
Crystallisation of membrane proteins mediated by antibody fragments.
X-ray structures of three different membrane proteins in complex with antibody fragments have been published. The binding of Fv or Fab fragments enlarges the hydrophilic part of integral membrane proteins, thereby providing additional surface for crystal contacts and space for the detergent micelle. In all reported cases, antibody binding was either essential for the crystallisation of the membrane protein or it substantially improved the diffraction quality of the crystals. Antibody-fragment-mediated crystallisation appears to be a valuable tool in particular for membrane proteins with very small hydrophilic or flexible domains.
Antibodies, Monoclonal↗
Protein structure: proton-pumping oxidases.
The crystal structures of two cytochrome c oxidases, one bacterial and one mammalian, offer insights into their roles in oxygen chemistry and as proton pumps.
Animals↗
Redox dependent conformational changes in the mixed valence form of the cytochrome c oxidase from p. The reorganization of glutamic acid 278 is coupled to the electron transfer from/to heme a and the binuclear center. denitrificans.
In this work we present the separation of FTIR difference signals induced by electron transfer to/from the redox centers of the cytochrome c oxidase from P. denitrificans and compare electrochemically induced FTIR difference spectra with those induced by CO photolysis. FTIR difference spectra of rebinding of CO to the half reduced (mixed valence) form of the cytochrome c oxidase after photolysis reflect the conformational changes induced by the rebinding of CO and by electron transfer reactions from heme a3 to heme a and further on to CUA. During this process, heme a3 (and CUB) are oxidized, whereas heme a and CuA are reduced. By subtracting these difference spectra from an electrochemically induced FTIR difference spectrum, where all four cofactors are reduced, the contributions for heme a3 (and CuB) could be separated. Correspondingly, the spectral contributions of heme a and CuA have been separated. The comparison of these spectra with the spectra calculated for the hemes on the basis of their redox dependent changes previously published in Hellwig et al., (Biochemistry 38, (1999) 1685-1694) show a high degree of similarity, except for additional signals coupled to the reorganization of the binuclear center upon CO rebinding. The separated spectra clearly show that the signals attributed to Glu278, an amino acid discussed to be crucial for proton pumping, is coupled to electron transfer to/from heme a and the binuclear heme a3-CuB center.
Electron Transport↗
Protein redox potential measurements based on kinetic analysis with mediated continuous-flow column electrolytic spectroelectrochemical technique. Application to TTQ-containing methylamine dehydrogenase.
Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectroelectrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TTQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.
Electrochemistry↗
Intermolecular electron transfer from substrate-reduced methylamine dehydrogenase to amicyanin is linked to proton transfer.
Within the methylamine dehydrogenase-amicyanin complex, intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) and copper. The ET reactions from two chemically distinct reduced forms of TTQ were studied. The quinol form of TTQ was generated by reduction by dithionite. An aminoquinol form of TTQ, in which an amino group displaces the carbonyl oxygen, was generated by reduction by the substrate methylamine. Thermodynamic analysis of the ET reactions suggested that the ET event was rate-limiting for the redox reaction between quinol TTQ and copper, but not for the ET reaction from aminoquinol TTQ to copper. Solvent kinetic isotope effect studies indicated that proton transfer was involved in the rate-limiting reaction step for the ET from the substrate-reduced enzyme, but not the dithionite-reduced enzyme. Solvent deuterium kinetic isotope effects of 1.5 and 12.2 were obtained, respectively, for the ET reactions from dithionite-reduced and substrate-reduced methylamine dehydrogenase. These results demonstrate that application of ET theory to the analysis of thermodynamic data for the intermolecular protein ET reactions can potentially be used to distinguish between true ET reactions and those which are gated or attenuated by adiabatic events. Kinetic models are presented to explain how the incorporation of the substrate-derived amino group into TTQ may alter the rate-limiting step for ET.
Bacterial Proteins↗
Kinetics of photooxidation of soluble cytochromes, HiPIP, and azurin by the photosynthetic reaction center of the purple phototrophic bacterium Rhodopseudomonas viridis.
The photosynthetic reaction center of Rhodopseudomonas viridis contains a bound tetraheme cytochrome c subunit which is the primary electron donor to the photooxidized special pair bacteriochlorophyll. We have tested a variety of soluble electron-transfer proteins for their ability to serve as secondary electron donors to the bacteriochlorophyll via the bound cytochrome by measuring the kinetics of reaction center heme reduction following photooxidation by a laser flash, as a function of soluble protein concentration and ionic strength. All of the soluble redox proteins utilized appear to interact with a negatively charged region on the reaction center and to transfer electrons to the 300-mV heme c-556 of the bound cytochrome. Rps. viridis cytochrome c2 was the best electron donor among those proteins tested, with a second-order rate constant extrapolated to infinite ionic strength of 1.2 x 10(6) M-1 s-1, which is two orders of magnitude larger than that of horse cytochrome c. Rps. viridis cytochrome c2 apparently binds to the reaction center at low ionic strength, as evidenced by a nonlinear dependence of kobs on protein concentration. The limiting first-order electron-transfer rate constant at 6 mM ionic strength is approximately 1300 s-1. Horse cytochrome c and the reaction center also form a complex, with a limiting first-order rate constant for electron transfer which is 5 times smaller than for cytochrome c2. Other cytochromes c2 are intermediate in reactivity. More distantly related cytochromes, HiPIP, and azurin are relatively poor electron donors under the conditions of assay.(ABSTRACT TRUNCATED AT 250 WORDS)
Azurin↗
Reactions of benzylamines with methylamine dehydrogenase. Evidence for a carbanionic reaction intermediate and reaction mechanism similar to eukaryotic quinoproteins.
It had been previously reported that aromatic amines were not substrates for the bacterial quinoprotein methylamine dehydrogenase. In this study, benzylamine-dependent activity was also not observed in the steady-state assay of this enzyme with the artificial electron acceptor phenazine ethosulfate (PES). Benzylamines did, however, stoichiometrically reduce the protein-bound tryptophan tryptophylquinone (TTQ) prosthetic group and acted as reversible competitive inhibitors of methylamine oxidation when the enzyme was assayed with PES. When methylamine dehydrogenase activity was monitored using a steady-state assay which employed its physiological electron acceptor amicyanin instead of PES, very low but detectable benzylamine-dependent activity was observed. The reactions of a series of para-substituted benzylamines with methylamine dehydrogenase were examined. A Hammett plot of the log of Ki values for the competitive inhibition by these amines against sigma p exhibited a negative slope. Rapid kinetic measurements allowed the determination of values of k3 and Ks for the reduction of TTQ by each of these amines. A Hammett plot of log k3 versus sigma p exhibited a positive slope, which suggests that the oxidation of these amines by methylamine dehydrogenase proceeds through a carbanionic reaction intermediate. A negative slope was observed for the correlation between log Ks and sigma p. Plots of log k3 and log Ks against substituent constants which reflected either resonance or field/inductive parameters for each para substituent indicated that the magnitude of k3 was primarily influenced by field/inductive effects while Ks was primarily influenced by resonance effects. No correlation was observed between either k3 or Ks and the relative hydrophobicity of the para-substituted benzylamines or steric parameters.(ABSTRACT TRUNCATED AT 250 WORDS)
Anions↗
Conversion of methylamine dehydrogenase to a long-chain amine dehydrogenase by mutagenesis of a single residue.
Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) dependent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalytic alpha subunit line a substrate channel that leads from the protein surface to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alphaPhe55 to alanine dramatically alters the substrate preference of MADH. The K(m) for methylamine increases from 9 microM to 15 mM. The preferred substrates are now primary amines with chain lengths of at least seven carbons. The K(m) for 1, 10-diaminodecane is 11 microM, compared to 1.2 mM for wild-type MADH. Despite the large variation in K(m) values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.
Alanine↗
Ionic strength dependence of the reaction between methanol dehydrogenase and cytochrome c-551i: evidence of conformationally coupled electron transfer.
The quinoprotein methanol dehydrogenase and cytochrome c-551i are two soluble acidic proteins that form a physiological complex in which electrons are transferred from pyrroloquinoline quinone to heme. The oxidation of methanol dehydrogenase by the cytochrome was studied as a function of ionic strength using stopped-flow spectroscopy. The dissociation constant (Kd) for complex formation decreased 2-fold with increasing ionic strength from 0.21 to 1.3 M and increased at higher ionic strengths. The rate constant for the electron transfer reaction (kET) increased 2-fold with increasing ionic strength from 0.21 to 1.3 M and decreased at higher ionic strengths. The variation of Kd and kET over this range of ionic strengths was described by Van Leeuwen theory, which takes into account monopole-dipole and dipole-dipole forces, in addition to the monopole-monopole force, to predict the interactions between large molecules. Analysis of the kinetic results in terms of these electrostatic interactions indicated the probable orientations for protein-protein binding and electron transfer. To explain the ionic strength dependence of the observed kET, a model is presented in which the true kET is reduced by a factor Kc, an equilibrium constant that describes some rearrangement of the proteins after a nonoptimal collision to produce the most efficient orientation for electron transfer. This model is consistent with the notion that the large reorganizational energy obtained from temperature-dependence studies of this electron transfer reaction [Harris, T. K., & Davidson, V. L. (1993) Biochemistry 32, 14145-14150] is due to such an intracomplex rearrangement.(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Oxidoreductases↗
Binding and electron transfer reactions between methanol dehydrogenase and its physiologic electron acceptor cytochrome c-551i: a kinetic and thermodynamic analysis.
The quinoprotein methanol dehydrogenase and cytochrome c-551i form a physiologic complex in which electrons are transferred from pyrroloquinoline quinone to heme. The reoxidation of methanol dehydrogenase by the cytochrome was studied by stopped-flow spectroscopy. The rate constant for the electron transfer reaction and the dissociation constant for complex formation were each determined at temperatures ranging from 20 to 50 degrees C. The electron transfer rates varied from 1.4 to 4.6 s-1. Analysis of the electron transfer reaction by Marcus theory yielded values of 1.9 eV for the reorganizational energy and 0.071 cm-1 for the electronic coupling and predicted a theoretical distance between redox centers of 15 A. Kinetically determined dissociation constants correlated well with a Kd of 375 microM which was determined in a direct ultrafiltration binding assay. Thermodynamic analysis of the dissociation constants indicated the importance of the hydrophobic effect in complex formation.
Alcohol Oxidoreductases↗
Electron-transfer reactions of photoreduced flavin analogues with c-type cytochromes: quantitation of steric and electrostatic factors.
We have found correlations between rate constants and the difference in redox potential of the reactants for electron-transfer reactions between oxidized cytochromes and either photoproduced riboflavin or flavin mononucleotide (FMN) semiquinones (the latter rate constants extrapolated to infinite ionic strength). The riboflavin-cytochrome rate constants are about 70% of those for reduction by lumiflavin, probably because of steric interference by the ribityl side chain. Reduction of cytochromes by FMN semiquinone was ionic strength dependent in all cases, due to electrostatic interactions. Extrapolation of rate constants to infinite ionic strength shows that the phosphate exerts a significant steric effect as well (rate constants average about 27% of those for lumiflavin, although part of this decrease is due to a difference in the semiquinone pK value). Differences in the magnitude of the FMN steric effect correlate well with surface topology differences for those cytochromes whose three-dimensional structures are known. Mitochondrial cytochromes c and the cytochromes c2 all showed attractive (plus-minus) interaction with FMN in spite of the fact that some of these proteins have large net negative charges. Four small c-type cytochromes (including Pseudomonas cytochrome c-551) show a weak repulsive interaction with FMN semiquinone. We conclude that flavosemiquinones interact at a site on the cytochromes that is near the exposed heme edge. There is a large positive electrostatic field at this site in mitochondrial cytochrome c and the cytochromes c2, but this region is primarily hydrophobic in Pseudomonas cytochrome c-551 and in the other small bacterial cytochromes.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer.
Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.
Bacterial Proteins↗
Reaction of carbon monoxide with the reduced active site of bacterial nitric oxide reductase.
Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.
Binding Sites↗
Proton and electron pathways in the bacterial nitric oxide reductase.
Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.
Bacterial Proteins↗