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H B Gray

Publications and source records attributed to H B Gray.

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

Electron transfer in ruthenium-modified proteins.

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)2(im)(His)2(+)-modified (bpy = 2,2'-bipyridine; im = imidazole) cytochrome c and azurin. A driving-force study with the His33 derivatives of cytochrome c indicates that the reorganization energy (lambda) for Fe2+-->Ru3+ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex, *Ru2+, or a reduced complex, Ru+, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe2+-->Ru3+ and Cu+-->Ru3+ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 A-1) that is consistent with prediction of the pathway model.

Azurin↗
PubMed1995

Rapid electron injection into multisite metalloproteins: intramolecular electron transfer in cytochrome oxidase.

The principles for the operation of redox-linked proton pumps are reviewed and applied to one specific pump, cytochrome oxidase. Systematic studies of internal electron transfer in the different redox states of this pump will be facilitated by the development of methods for rapid electron injection into the metal centers of the enzyme. Two methods that have been employed to generate electron donors are pulse radiolysis and laser flash photolysis. The rate of electron injection from photoexcited Ru-modified cytochrome c or triplet Zn-cytochrome c into the CuA center is about 10(5) s-1, and the CuA/cytochrome a electron equilibration rate is 2 x 10(4) s-1. Electron transfer from cytochrome a to the cytochrome a3-CuB site occurs at 2 x 10(5) s-1 in the half-reduced enzyme, whereas the rate is only 2 x 10(2) s-1 in the peroxide intermediate, despite a much higher driving force. It is likely that variations in distant electronic coupling attributable to a ligand shuttle, as well as changes in the reorganization energy of one or more of the redox centers, contribute to the control of internal electron flow in the enzyme.

Amino Acid Sequence↗
PubMed1995

pH-dependent equilibria of yeast Met80Ala-iso-1-cytochrome c probed by NMR spectroscopy: a comparison with the wild-type protein.

BACKGROUND: Cytochrome c has five distinct pH-dependent conformational states, including two alkaline forms of unknown structure. It is believed that in both of the alkaline forms a Lys residue is ligated to the heme, but the identity of the Lys residue is different. Exchange between these forms would require extensive structural rearrangement. Mutation of the heme axial ligand (Met80) to Ala in Saccharomyces cerevisiae iso-1-cytochrome c yields a protein (Ala80cyt c) capable of binding exogenous ligands such as dioxygen and cyanide. We have analyzed the 1H NMR spectra of this mutant at various pH values in the hope of gaining insight into the structure of the acidic and alkaline forms of native cytochrome c. RESULTS: The pH dependence of the 1H NMR spectrum of ferriAla80cyt c is consistent with the high-spin/low-spin transition (pKa = 6.5) observed by absorption spectroscopy. The T1 values for the low-spin form are consistent with OH ligation, as inferred previously from absorption and electron paramagnetic resonance spectroscopic results. The pH-dependent equilibria of ferriAla80cyt c differ from those of the wild-type protein. Both Ala80 and wild-type ferricyt c appear to have the same iron coordination at low pH (approximately equal to 2), while only one alkaline form of Ala80cyt c (versus two for WTcyt c) was detected. CONCLUSIONS: The differences between the pH dependence of the 1H NMR spectra of Ala80cyt c and those of the wild-type protein demonstrate that the heme axial ligands influence the relative energies of the conformational states of cytochrome c. The results are consistent with the notion that a large rearrangement is required to switch between the two alkaline forms.

Cytochrome c Group↗
PubMed1995

Electron tunneling in azurin: the coupling across a beta-sheet.

BACKGROUND: We would like to understand how electron flow is controlled in biological molecules. Standard theories calculate the rate for long distance electron transfer (ET) as the product of electronic coupling (the square of the electron tunneling matrix element) and nuclear (Franck-Condon) factors. Much attention has been directed to the role of protein secondary and tertiary structure in the tunneling coupling, focusing on the interplay between different types of chemical bonds. Here we have evaluated the relative contributions of covalent bonds, hydrogen bonds and through-space jumps in coupling through a beta-strand or across a beta-sheet section of a blue copper protein, azurin. RESULTS: We have analyzed four distant electronic couplings in azurin. Each coupling is between the copper atom and a Ru(bpy)2(im) complex attached to a histidine on the protein surface. In three experiments the intervening medium was a simple beta-strand, while in the fourth experiment it was a section of beta-sheet. CONCLUSIONS: We have shown that electron tunneling in a protein can be broken down into ET 'tubes' of pathways through specific covalent and hydrogen bonds. These ET tubes encapsulate trivial interference effects and can expose crucial inter-tube interference effects. In coupling through a beta-sheet, hydrogen bonds are as important as covalent links, and are the primary source for tube interference.

Azurin↗
PubMed1994

Combined pH gradient and anion-exchange high-performance liquid chromatographic separation of oligodeoxyribonucleotides.

A novel method of elution using a pH gradient to separate small thymidine- and guanosine-containing oligonucleotides on a Pharmacia Mono Q HR anion-exchange column is described. The method is based on the alkaline titration of ring protons of the thymine and guanine base moieties and results in excellent separations of di-, tri- and tetranucleotides that either are not resolved in salt gradients near neutral pH or require long elution times when salt gradient elution is used with strongly alkaline eluents.

Anion Exchange Resins↗
PubMed1994

Mechanism of catalytic oxygenation of alkanes by halogenated iron porphyrins.

Halogenation of an iron porphyrin causes severe saddling of the macrocyclic structure and a large positive shift in the iron(III)/(II) redox couple. Although pre-halogenated iron(II) porphyrins such as Fe(TFPPBr8) [H2TFPPBr8, beta-octabromo-tetrakis(pentafluorophenyl)-porphyrin] are relatively resistant to autoxidation, they rapidly reduce alkyl hydroperoxides. These and related reactivity studies suggest that catalysis of alkane oxygenation by Fe(TFPPBr8)Cl occurs through a radical-chain mechanism in which the radicals are generated by oxidation and reduction of alkyl hydroperoxides.

Alkanes↗
PubMed1994

Electron transfer in cytochrome c depends upon the structure of the intervening medium.

BACKGROUND: Long-distance electron-transfer (ET) reactions through proteins are involved in a great many biochemical processes; however, the way in which the protein structure influences the rates of these reactions is not well understood. We have therefore measured the rates of intramolecular ET from the ferroheme to a bis(2,2'-bipyridine)imidazoleruthenium(III) acceptor at histidine 39 or 54 in derivatives of yeast iso-1-cytochrome c, and studied the effect of an asparagine to isoleucine mutation at position 52, a residue situated between the heme and the electron acceptor. RESULTS: The Fe2+-->Ru3+ rate constants demonstrate that residue 52 affects ET from the heme to His54 (Ile52 > Asn52), but not to His39 (Ile52 = Asn52). The enhanced Fe(2+)-Ru3+(His54) electronic coupling for the N52I/K54H protein is in good agreement with sigma-tunneling calculations, which predict the length of the ET pathways between the heme and His54. CONCLUSION: The structure of the intervening medium between the heme and electron acceptors at the protein surface influences the donor-acceptor couplings in cytochrome c.

Base Sequence↗
PubMed1993

Structurally engineered cytochromes with unusual ligand-binding properties: expression of Saccharomyces cerevisiae Met-80-->Ala iso-1-cytochrome c.

A strategy has been developed to express and purify a recombinant, nonfunctional axial-ligand mutant of iso-1-cytochrome c (Met-80-->Ala) in Saccharomyces cerevisiae in quantities necessary for extensive biophysical characterization. It involves coexpressing in the same plasmid (YEp213) the nonfunctional gene with a functional gene copy for complementation in a selective medium. The functional gene encodes a product with an engineered metal-chelating dihistidine site (His-39 and Leu-58-->His) that enables efficient separation of the two isoforms by immobilized metal-affinity chromatography. The purified Met-80-->Ala protein possesses a binding site for dioxygen and other exogenous ligands. Absorption spectra of several derivatives of this mutant show striking similarities to those of corresponding derivatives of horseradish peroxidase, myoglobin, and cytochrome P450. The use of a dual-gene vector for cytochrome c expression together with metal-affinity separation opens the way for the engineering of variants with dramatically altered structural and catalytic properties.

Alanine↗
PubMed1993

Evidence for a salt-induced conformational transition in UV-irradiated superhelical PM2 DNA.

Upon treatment with UV irradiation, native (supercoiled) PM2 DNA undergoes an increase in electrophoretic mobility relative to the nicked circular form in the presence of 1 M NaCl or 5 mM CaCl2 or MgCl2. This effect is dependent upon supercoiling in that the relative electrophoretic mobility decreases with decreasing superhelical density of the molecule. These findings indicate that supercoil-dependent aspects of the secondary and tertiary structure of nonirradiated PM2 DNA can be altered by a combination of UV irradiation and any of the ionic environments above. We show that the alteration is not the result of a conversion of Z-DNA segments to a right-handed helix or to a renaturation of denatured regions in PM2 DNA. Circular dichroism studies do not support a simple model in which A-form DNA induced by superhelical stress is converted to B-form DNA by UV-induced photodamage and salt. We, therefore, present three alternative explanations for these observations two of which invoke conformational transitions in secondary structure and a third which requires a change in tertiary structure due to an increase in flexibility.

Bacteriophages↗
PubMed1993

Site saturation of the histidine-46 position in Pseudomonas aeruginosa azurin: characterization of the His46Asp copper and cobalt proteins.

Cassette mutagenesis has been used to replace the copper ligand His46 of Pseudomonas aeruginosa azurin with 19 other amino acids and a stop codon. Several mutant proteins were expressed in Escherichia coli and isolated; however, only the variant in which His was replaced by Asp exhibited the spectral characteristics of a blue (type 1) center. The spectroscopic and electrochemical properties of this mutant protein show that the copper site is perturbed relative to wild-type azurin. The absorption spectrum of Cu(II)(His46Asp) azurin exhibits a S(Cys)-->Cu(II) band at 612 nm, as well as weaker features at approximately 300, 454, and approximately 850 nm; its EPR spectrum is rhombic (g parallel = 2.327(1), gx approximately 2.03, and gy approximately 2.07; A parallel = 22(2) x 10(-4), Ax approximately 46 x 10(-4), and Ay approximately 22 x 10(-4) cm-1). The reduction potential of the mutant (260 mV vs NHE at pH 8.5; 297 mV at pH 5.0) is lower than that of wild-type azurin (288 mV at pH 8.5; 349 mV at pH 5.0). The S(Cys)-->Co(II) absorption bands (approximately 300 and 362 nm) in Co(II)(His46Asp) azurin are strongly blue-shifted relative to those (330 and 375 nm) in the spectrum of the Co(II) (His46) protein, whereas the intensities of the ligand-field bands in the 500-650-nm region (epsilon approximately 100 M-1 cm-1) indicate a five-coordinate Co(II) environment.

Azurin↗
PubMed1993

Identification of chemical modification sites on metalloproteins by capillary electrophoresis.

Capillary electrophoresis (CE) has been used to separate the peptides obtained from tryptic digestion of ruthenium-modified cytochrome c. The modified peptide was identified from a comparison of the elution profile and absorbance characteristics of the native and modified proteins. Automatic fraction collection on the CE instrument provides sufficient amounts of this modified peptide for amino acid sequencing. Capillary electrophoresis has also been used to monitor the modification reaction and to optimize the modification efficiency. The methodologies have been extended to myoglobin in order to monitor the modification of multiple surface sites.

Animals↗
PubMed1991

Vectors containing infrequently cleaved restriction sites for use in BAL 31 nuclease-assisted and end-label-mediated analysis of cloned DNA fragments.

Cloning vectors derived from plasmids pUC8 and pUC18 and phage M13mp10 were constructed so as to have multiple cloning sites (MCS) flanked by the recognition/cleavage sites for the Sfi I and Not I restriction nucleases. Cleavage of vectors containing cloned DNA fragments with either of the infrequently cleaving Sfi I or Not I endonucleases will usually yield linear DNAs cleaved only at the corresponding site in the MCS, so that the cloned insert can be degraded unidirectionally by the duplex exonuclease activity of the BAL 31 nucleases until an amount equal to the length of the vector has been degraded. The ends of the above constructs resulting from cleavage with Not I or Sfi I can readily be labeled, with labeling at only the terminus of the cloned DNA available for the Sfi I site. The BAL 31 nuclease-mediated procedures enhance a previous technique for mapping of restriction enzyme fragments, allow for localization of sequences in cloned segments for which a probe is available, and improve a method for sequencing cloned inserts through the production of sets of nested unidirectional deletions from either end of the parent cloned fragment. The advantages of end-label-mediated restriction site mapping using the above vectors over existing such procedures are also demonstrated.

Bacteriophages↗
PubMed1990

Mechanism of exonuclease action of BAL 31 nuclease.

Two kinetically and molecularly distinct forms ('fast' (F) and 'slow' (S] of nuclease BAL 31 from Alteromonas espejiana effect the length reduction of linear duplex DNAs through a 3'----5'-directed exonuclease activity in conjunction with an endonuclease activity against the 5'-terminated single-stranded tails generated by the exonuclease activity. No evidence for a 5'----3' mode of exonuclease action was seen. Single-stranded DNA is degraded predominantly by the 3'----5' exonuclease action. There is a pronounced decrease, to roughly constant values, of the average lengths of the tails in partially digested duplexes at a constant extent of digestion with increasing nuclease concentration. This decrease correlates with an increasing extent of ligatability, in the absence of repair, under conditions favoring the joining of fully base-paired ends. The exonuclease action, at least against duplex substrates, is quasi-processive and removes approx. 18 and 28 nucleotides per productive enzyme-substrate encounter for the S and F species, respectively. The dependence on Ca2+ and Mg2+ concentrations of the activities has been determined.

Bacteriophages↗
PubMed1990

Precursor-product relationship of larger to smaller molecular forms of the BAL 31 nuclease from Alteromonas espejiana: preferential removal of duplex exonuclease relative to endonuclease activity by proteolysis.

Two molecularly and kinetically distinct major species of the extracellular nuclease BAL 31 from Alteromonas espejiana, previously characterized as the "fast" (F) and "slow" (S) BAL 31 nucleases, have been evidenced to derive from proteolysis starting from a still larger (approximately 120 kDa) precursor nuclease. The expected protease activity in the culture fluid has been confirmed and is strongly dependent on the cell growth phase. The disappearance of the largest nuclease species with the concomitant sequential appearance of first the F and then the S species has been demonstrated for nuclease obtained from culture supernatants as a function of cell growth phase. Nuclease from periplasmic extracts displayed very little of the F and S nucleases. Treatment of purified F nuclease with Pronase or subtilisin readily converted it to species with only a few percent of the native exonuclease activity against duplex DNA but retaining much of the initial activity against single-stranded DNA. Electrophoresis in nuclease-detecting gels demonstrated a parallel conversion of the larger species to one indistinguishable in molecular weight from the S species. The observed loss of exonuclease activity could correspond to the conversion of the F to the S nuclease. However, treatment of S nuclease with subtilisin resulted in a drastic reduction of exonuclease activity of this enzyme on duplex DNA with retention of most of the activity against single-stranded and nicked circular duplex DNA substrates. Evidence of internal proteolysis of the S nuclease could be seen after electrophoresis in denaturing gels but only after the denaturation buffer was adjusted to 6 M in urea. The preferential removal of the exonuclease activity may enhance the usefulness of the BAL 31 nuclease in such applications as heteroduplex mapping.

Blotting, Western↗
PubMed1989

1H NMR characterization of Chromatium gracile high-potential iron protein and its ruthenium-modified derivatives. Modulation of the reduction potentials in low- and high-potential [Fe4S4] ferredoxins.

The NMR spectra of the high-potential iron protein from the photosynthetic bacterium Chromatium gracile and its ruthenium-labeled (His-42 and His-20) derivatives are reported. The isotropically shifted resonances in both the oxidized and reduced forms show a complex pH dependence due to the presence of three ionizable residues (Glu-44, His-20, and His-42). Assignments have been made to specific residues and the spectral features compared to those of other bacterial HiPIP's. The decrease in the reduction potential with increasing pH for this class of proteins is attributed to stabilization of the oxidized state of the cluster by delocalization of electron density onto the neighboring Tyr-19 residue.

Bacterial Proteins↗