Biomedical subjects

Harry B Gray

Publications and source records attributed to Harry B Gray.

At least 55 records · Page 3Linked to original sources

PubMed2004

Oxoiron(IV) in chloroperoxidase compound II is basic: implications for P450 chemistry.

With the use of x-ray absorption spectroscopy, we have found that the Fe-O bond in chloroperoxidase compound II (CPO-II) is much longer than expected for an oxoiron(IV) (ferryl) unit; notably, the experimentally determined bond length of 1.82(1) A accords closely with density functional calculations on a protonated ferryl (Fe(IV)-OH, 1.81 A). The basicity of the CPO-II ferryl [pKa > 8.2 (where Ka is the acid dissociation constant)] is attributable to strong electron donation by the axial thiolate. We suggest that the CPO-II protonated ferryl is a good model for the rebound intermediate in the P450 oxygenation cycle;with elevated pKa values after one-electron reduction, thiolate-ligated ferryl radicals are competent to oxygenate saturated hydrocarbons at potentials that can be tolerated by folded polypeptide hosts.

Chemical Phenomena↗

PubMed2004

X-ray magnetic circular dichroism of Pseudomonas aeruginosa nickel(II) azurin.

We show that X-ray magnetic circular dichroism (XMCD) can be employed to probe the oxidation states and other electronic structural features of nickel active sites in proteins. As a calibration standard, we have measured XMCD and X-ray absorption (XAS) spectra for the nickel(II) derivative of Pseudomonas aeruginosa azurin (NiAz). Our analysis of these spectra confirms that the electronic ground state of NiAz is high-spin (S = 1); we also find that the L(3)-centroid energy is 853.1(1) eV, the branching ratio is 0.722(4), and the magnetic moment is 1.9(4) mu(B). Density functional theory (DFT) calculations on model NiAz structures establish that orbitals 3d(x2-y2) and 3d(z2) are the two valence holes in the high-spin Ni(II) ground state, and in accord with the experimentally determined orbital magnetic moment, the DFT results also demonstrate that both holes are highly delocalized, with 3d(x2-y2) having much greater ligand character.

Azurin↗

PubMed2004

Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins.

Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins. Cu(I) to "Re(II)" electron tunneling in Re(H107) azurin occurs in the microsecond range. This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep ("hopping") mechanism might be involved. Although a Y108 radical can be generated by flash-quenching a Re(H107)M(II) (M=Cu, Zn) protein, the evidence suggests that it is not an active intermediate in the enhanced Cu(I) oxidation. Rather, the likely explanation is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radical), followed by electron tunneling from Cu(I) to the hole in the imidazole ligand.

Azurin↗

PubMed2004

Photoswitchable luminescence of rhenium(i) tricarbonyl diimines.

The synthesis, characterization, and X-ray crystal structures of [Re(diimine)(CO)(3)(dpe)](PF(6)) (dpe = 1,2-di(4-pyridyl)ethylene) compounds are reported. The cis-dpe complexes exhibit yellow luminescence after UV excitation, whereas the trans-dpe counterparts are nonluminescent. The luminescence quantum yields of the cis-dpe complexes are strongly dependent on the identity of the diimine ligand. Irradiation (350 nm) of the trans-dpe complexes induces trans --> cis dpe-ligand isomerization with quantum yields on the order of 0.2, and this process leads to an on-switching of yellow luminescence. After long 350-nm irradiation times, a steady state composed of roughly 70% cis- and 30% trans-dpe complexes is reached. The reverse cis --> trans photoisomerization reaction is induced by irradiating the cis-dpe complexes at 250 nm, switching off the yellow luminescence. For 250-nm excitation, photodecomposition of the [Re(diimine)(CO)(3)(dpe)](+) complexes competes efficiently with photoisomerization.

Journal Article↗

PubMed2004

NMR-validated structural model for oxidized Rhodopseudomonas palustris cytochrome c(556).

The structure of oxidized Rhodopseudomonas palustris cytochrome c(556) has been modeled after that of high-spin cytochrome c' from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.

Algorithms↗

PubMed2004

Alpha-synuclein: stable compact and extended monomeric structures and pH dependence of dimer formation.

The protein alpha-synuclein, implicated in Parkinson's disease, was studied by combining nano-electrospray ionization (N-ESI) mass spectrometry and ion mobility. It was found that both the charge-state distribution in the mass spectra and the average protein shape deduced from ion mobility data, depend on the pH of the spray solution. Negative-ion N-ESI of pH 7 solutions yielded a broad charge-state distribution from -6 to -16, centered at -11, and ion mobility data consistent with extended protein structures. Data obtained for pH 2.5 solutions, on the other hand, showed a narrow charge-state distribution from -6 to -11, centered at -8, and ion mobilities in agreement with compact alpha-synuclein structures. The data indicated that there are two distinct families of structures: one consisting of relatively compact proteins with eight or less negative charges and one consisting of relatively extended structures with nine or more charges. The average cross section of a-synuclein at pH 2.5 is 33% smaller than for the extended protein sprayed from pH 7 solution. Significant dimer formation was observed when sprayed from pH 7 solution but no dimers were observed from the low pH solution. A plausible mechanism for aggregate formation in solution is proposed.

Amino Acid Sequence↗

PubMed2004

The stability of the cytochrome c scaffold as revealed by NMR spectroscopy.

NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions. The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein. The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c. The presence of denaturants decreases the temperature at which the native Met80 axial ligand is displaced from the iron center under the present mild alkaline conditions. The process can be described in terms of protein fragments behaving as unfolding units of different stability. The comparison between the two proteins indicates that the loop+helix connecting the proximal and distal sites, as well as the long Met80-containing loop immediately after a short helix, are structural characteristics of mitochondrial cytochrome c that appear to be responsible for the Met80-iron(III) bond fragility.

Animals↗

PubMed2004

Cobaltocene-mediated catalytic monooxygenation using holo and heme domain cytochrome P450 BM3.

The feasibility of replacing NADPH with 1,1'-dicarboxycobaltocene in the catalytic cycle of cytochrome P450 BM3 has been explored. Using the holoprotein, the surrogate mediator was observed to reduce both the FAD and FMN in the reductase domain, as well as the iron in the heme domain. In an electrochemical system, the mediator was able to support lauric acid hydroxylation at a rate of 16.5 nmol product/nmol enzyme/minute. Similar electron transfer and catalysis were observed for the heme domain alone in the presence of the metallocene; the turnover rate in this case was 1.8 nmol product/nmol enzyme/minute. Parallel studies under the same conditions using a previously reported cobalt sepulchrate mediator showed that the two systems give similar results for both the holoenzyme and the heme domain.

Bacillus megaterium↗

PubMed2003

Spectroscopy and reactivity of a photogenerated tryptophan radical in a structurally defined protein environment.

Near-UV irradiation of structurally characterized [Re(I)(CO)3(1,10-phenanthroline)(Q107H)](W48F/Y72F/H83Q/Y108W)AzM(II) [Az = Pseudomonas aeruginosa azurin, M = Cu, Zn]/[Co(NH3)5Cl]Cl2 produces a tryptophan radical (W108*) with unprecedented kinetic stability. After rapid formation (k = 2.8 x 106 s-1), the radical persists for more than 5 h at room temperature in the folded ReAzM(II) structure. The absorption spectrum of ReAz(W108*)M(II) exhibits maxima at 512 and 536 nm. Oxidation of K4[Mo(CN)8] by ReAz(W108*)Zn(II) places the W108*/W108 reduction potential in the protein above 0.8 V vs NHE.

Azurin↗

PubMed2003

Nanosecond photoreduction of cytochrome p450cam by channel-specific Ru-diimine electron tunneling wires.

We report the synthesis and characterization of Ru-diimine complexes designed to bind to cytochrome p450cam (CYP101). The sensitizer core has the structure [Ru(L(2))L'](2+), where L' is a perfluorinated biphenyl bridge (F(8)bp) connecting 4,4'-dimethylbipyridine to an enzyme substrate (adamantane, F(8)bp-Ad), a heme ligand (imidazole, F(8)bp-Im), or F (F(9)bp). The electron-transfer (ET) driving force (-deltaG degrees ) is varied by replacing the ancillary 2,2'-bipyridine ligands with 4,4',5,5'-tetramethylbipyridine (tmRu). The four complexes all bind p450cam tightly: Ru-F(8)bp-Ad (1, K(d) = 0.077 microM); Ru-F(8)bp-Im (2, K(d) = 3.7 microM); tmRu-F(9)bp (3, K(d) = 2.1 microM); and tmRu-F(8)bp-Im (4, K(d) = 0.48 microM). Binding is predominantly driven by hydrophobic interactions between the Ru-diimine wires and the substrate access channel. With Ru-F(8)bp wires, redox reactions can be triggered on the nanosecond time scale. Ru-wire 2, which ligates the heme iron, shows a small amount of transient heme photoreduction (ca. 30%), whereas the transient photoreduction yield for 4 is 76%. Forward ET with 4 occurs in roughly 40 ns (k(f) = 2.8 x 10(7) s(-)(1)), and back ET (Fe(II) --> Ru(III), k(b) approximately 1.7 x 10(8) s(-)(1)) is near the coupling-limited rate (k(max)). Direct photoreduction was not observed for 1 or 3. The large variation in ET rates among the Ru-diimine:p450 conjugates strongly supports a through-bond model of Ru-heme electronic coupling.

Camphor 5-Monooxygenase↗

PubMed2003

Gold electrodes wired for coupling with the deeply buried active site of Arthrobacter globiformis amine oxidase.

Diethylaniline-terminated oligo(phenyl-ethynyl)-thiol (DEA-OPE-SH) wires on Au-bead electrodes facilitate electron tunneling to and from the deeply buried topaquinone (TPQ) cofactor in Arthrobacter globiformis amine oxidase (AGAO). Reversible cyclic voltammograms were observed when AGAO was adsorbed onto this DEA-OPE-SAu surface: the 2e-/2H+ reduction potential is -140 mV versus SCE.

Amine Oxidase (Copper-Containing)↗

PubMed2003

Biological inorganic chemistry at the beginning of the 21st century.

Advances in bioinorganic chemistry since the 1970s have been driven by three factors: rapid determination of high-resolution structures of proteins and other biomolecules, utilization of powerful spectroscopic tools for studies of both structures and dynamics, and the widespread use of macromolecular engineering to create new biologically relevant structures. Today, very large molecules can be manipulated at will, with the result that certain proteins and nucleic acids themselves have become versatile model systems for elucidating biological function.

Chemistry, Bioinorganic↗

PubMed2003

The protein-folding speed limit: intrachain diffusion times set by electron-transfer rates in denatured Ru(NH3)5(His-33)-Zn-cytochrome c.

The kinetics of electron transfer from the triplet-excited Zn-porphyrin to a Ru(NH(3))(5)(His-33)(3+) complex have been measured in Zn-substituted ruthenium-modified cytochrome c under denaturing conditions. In the folded protein, the electron-tunneling rate constant is 7.5 x 10(5) s(-1). As the protein is denatured with guanidine hydrochloride, a faster adiabatic electron-transfer reaction appears (4.0 x 10(6) s(-1), [guanidine hydrochloride] = 5.4 M) that is limited by the rate of intrachain diffusion to bring the Zn-porphyrin and Ru complex into contact. The 250-ns contact time for formation of a 15-residue loop in denatured cytochrome c is in accord with a statistical model developed by Camacho and Thirumalai [Camacho, C. J. & Thirumalai, D. (1995) Proc. Natl. Acad. Sci. USA 92, 1277-1281] that predicts that the most probable transient loops formed in denatured proteins are comprised of 10 amino acids. Extrapolation of the cytochrome c contact time to a 10-residue loop sets the folding speed limit at approximately 10(7) s(-1).

Cytochrome c Group↗

PubMed2003

Structural model for an alkaline form of ferricytochrome C.

An (15)N-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximately equal to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOEs for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5 +/- 0.2 A. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.

Cytochromes c↗

PubMed2003

Aerobic oxidations catalyzed by chromium corroles.

Oxochromium(V) complexes of 5,10,15-tris(pentafluorophenyl)corrole and brominated derivatives oxygenate substrates (triphenylphosphine and norbornene) with concomitant production of chromium(III). Regeneration of CrVO by reaction of dioxygen with CrIII completes an aerobic catalytic cycle, with very large solvent effects; in acetonitrile, rapid initial turnovers observed initially are shut down by formation of CrIVO, while in toluene, THF, and methanol, relatively slow reactions are further inhibited by product formation.

Aerobiosis↗

PubMed2003

Cloning, heterologous expression, and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris.

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).

Amino Acid Sequence↗

PubMed2003

15N-1H Residual dipolar coupling analysis of native and alkaline-K79A Saccharomyces cerevisiae cytochrome c.

Residual dipolar couplings (RDCs) and pseudocontact shifts are experimentally accessible properties in nuclear magnetic resonance that are related to structural parameters and to the magnetic susceptibility anisotropy. We have determined RDCs due to field-induced orientation of oxidized-K79A and reduced cytochrome c at pH 7.0 and oxidized-K79A cytochrome c at pH 11.1 through measurements of amide (15)N-(1)H (1)J couplings at 800 and 500 MHz. The pH 7.0 RDCs for Fe(III)- and Fe(II)-cytochrome c together with available nuclear Overhauser effects were used to recalculate solution structures that were consistent with both sets of constraints. Molecular magnetic susceptibility anisotropy values were calculated for both redox states of the protein. By subtracting the residual dipolar couplings (RDCs) of the reduced form from those of the oxidized form measured at the same magnetic field (800 MHz), we found the RDC contribution of the paramagnetic metal ion in the oxidized protein. The magnetic susceptibility anisotropy, which was calculated from the structure, was found to be the same as that of the paramagnetic metal ion obtained independently from pseudocontact shifts, thereby indicating that the elements of secondary structure either are rigid or display the same mobility in both oxidation states. The residual dipolar coupling values of the alkaline-K79A form are small with respect to those of oxidized native cytochrome, whereas the pseudocontact shifts are essentially of the same magnitude, indicating local mobility. Importantly, this is the first time that mobility has been found through comparison of RDCs with pseudocontact shifts.

Anisotropy↗

PubMed2003

Electron tunneling through proteins.

Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these 'currents of life'. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates. We also discuss electron transfer across protein-protein interfaces that has been probed both in solution and in structurally characterized crystals. It is now clear that electrons tunnel between sites in biological redox chains, and that protein structures tune thermodynamic properties and electronic coupling interactions to facilitate these reactions. Our work has produced an experimentally validated timetable for electron tunneling across specified distances in proteins. Many electron tunneling rates in cytochrome c oxidase and photosynthetic reaction centers agree well with timetable predictions, indicating that the natural reactions are highly optimized, both in terms of thermodynamics and electronic coupling. The rates of some reactions, however, significantly exceed timetable predictions: it is likely that multistep tunneling is responsible for these anomalously rapid charge transfer events.

Azurin↗