PubMed Health⌕ Search

Biomedical subjects

Russ Hille

Publications and source records attributed to Russ Hille.

29 records · Page 2Linked to original sources
PubMed2002

The reaction mechanism of xanthine oxidase: evidence for two-electron chemistry rather than sequential one-electron steps.

Current research on xanthine oxidase has favored a mechanism involving base-catalyzed proton abstraction from a Mo-OH group, allowing nucleophilic attack on the substrate and hydride transfer from the substrate to Mo=S group in the active site. During the course of this reaction mechanism, the molybdenum redox cycles from MoVI to MoIV, with reoxidation of the MoIV speices to form the EPR active MoV intermediate. However, it has also been suggested that the reaction occurs in two subsequent one-electron steps. We have determined kinetic parameters kred and kred/Kd for a variety of plausible substrates as well as the one-electron reduction potentials for these substrates. Our data indicate no correlation between these kinetic parameters and their one-electron reduction potentials, as would be expected if the enzyme were using two subsequent one-electron reduction steps. Our results provide additional support to current evidence for the favored two-electron reduction mechanism.

Electrons↗
PubMed2002

The active site of arsenite oxidase from Alcaligenes faecalis.

Arsenite oxidase, a member of the DMSO reductase family of molybdenum enzymes, has two molecules of guanosine dinucleotide molybdenum cofactor coordinating the molybdenum at the active site. X-ray absorption spectroscopy indicates that the Mo-S bonds shorten from 2.47 to 2.37 A upon reduction with the physiological substrate. It also indicates the presence of an oxo ligand at 1.70 A in both oxidized and reduced forms of the enzyme, together with a short, 1.83 A, Mo-O bond in the oxidized form that is lost upon reduction. Resonance Raman spectroscopy indicates that the two pterin dithiolene moieties have different aromaticities, with one, the Q-pterin, having a more discrete dithiolate structure while the other, the P-pterin, has considerable pi-delocalization. Our results indicate that the structure of arsenite oxidase is intermediate between that seen in other molybdenum enzymes, in which one ligand to the metal is provided by the polypeptide (serine, cysteine, or selenocysteine), and tungsten enzymes that lack a peptide ligand.

Alcaligenes↗
PubMed2002

Oxo, sulfido, and tellurido Mo-enedithiolate models for xanthine oxidase: understanding the basis of enzyme reactivity.

The active site of the mononuclear molybdenum enzyme xanthine oxidase has an LMoOS(OH) center that catalyzes the hydroxylation of substrate (L representing an enedithiolate ligand contributed by a pterin cofactor in the enzyme). Reaction of the enzyme with cyanide results in the replacement of the Mo=S group with a second Mo=O group, which results in loss of enzyme activity. To understand the basis for this loss of activity, we have computationally examined the interaction of a model for the LMoO2(OH) as well the LMoOTe(OH) congener of the enzyme with formamide (a substrate for the enzyme). Our electronic structure calculations for the oxo congener indicate a reduced electron density on the hydrogen being transferred from substrate in the course of the reaction, a shorter O-H bond in the transition state, and a longer nascent O-C bond of product, factors which combine to account for the loss of reactivity in the LMoO2(OH) species. Interestingly, our calculations indicate that the Te congener is characterized by an increased electron density on the hydrogen species being transferred, a longer Te-H bond in the transition state, and a shorter O-C nascent bond in the product and suggest that a Te congener of xanthine oxidase, were it to be prepared experimentally, should exhibit catalytic activity.

Molecular Mimicry↗
PubMed2002

Functional asymmetry of photosystem II D1 and D2 peripheral chlorophyll mutants of Chlamydomonas reinhardtii.

The peripheral accessory chlorophylls (Chls) of the photosystem II (PSII) reaction center (RC) are coordinated by a pair of symmetry-related histidine residues (D1-H118 and D2-H117). These Chls participate in energy transfer from the proximal antennae complexes (CP43 and CP47) to the RC core chromophores. In addition, one or both of the peripheral Chls are redox-active and participate in a low-quantum-yield electron transfer cycle around PSII. We demonstrate that conservative mutations of the D2-H117 residue result in decreased Chl fluorescence quenching efficiency attributed to reduced accumulation of the peripheral accessory Chl cation, Chl(Z)(+). In contrast, identical symmetry-related mutations at residue D1-H118 had no effect on Chl fluorescence yield or quenching kinetics. Mutagenesis of the D2-H117 residue also altered the line width of the Chl(Z)(+) EPR signal, but the line shape of the D1-H118Q mutant remained unchanged. The D1-H118 and D2-H117 mutations also altered energy transfer properties in PSII RCs. Unlike wild type or the D1-H118Q mutant, D2-H117N RCs exhibited a reduced CD doublet in the red region of Chl absorbance band, indicative of reduced energetic coupling between P680 and the peripheral accessory Chl. In addition, transient absorption measurements of D2-H117N RCs, excited on the blue side of the Chl absorbance band, exhibited a ( approximately 400 fs) pheophytin Q(X) band bleach lifetime component not seen in wild-type or D1-H118Q RCs. The origin of this component may be related to delayed fast-energy equilibration of the excited state between the core pigments of this mutant.

Absorption↗
PubMed2002

Iron-sulfur cluster biosynthesis: characterization of Schizosaccharomyces pombe Isa1.

Eukaryotic Isa1 is one of several mitochondrial proteins that have been implicated in Fe-S cluster assembly paths in vivo. We report the first biochemical characterization of an eukaryotic member of this family and discuss this in the context of results from in vivo studies and studies of bacterial homologues. Schizosaccharomyces pombe Isa1 is a multimeric protein carrying [2Fe-2S](2+) clusters that have been characterized by Mössbauer and optical spectroscopic studies. Complex formation with a redox-active ferredoxin has been identified through crosslinking experiments and the coordination chemistry and stability of the native clusters has been investigated through site-directed mutagenesis and spectroscopic analysis. Electronic supplementary material to this paper, containing Mössbauer and UV-visible spectra for mutant Isa1 proteins, can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0330-2.

Alanine↗
PubMed2002

Crystal structure and stability studies of C77S HiPIP: a serine ligated [4Fe-4S] cluster.

The crystal structure of Chromatium vinosum C77S HiPIP has been determined and is compared with that of wild type. This is the first reported crystal structure of a Ser ligated [4Fe-4S] cluster and reveals a 0.11 A shortening of the Fe-O bond (relative to Fe-S), but only minor structural alterations of the overall tertiary structure. Coordination changes are corroborated by resonance Raman spectroscopy. Comparison of the crystal and solution structures for HiPIPs identifies Phe48 as the main controller of solvent access to the Fe-S cluster; however, there is no significant change in cluster solvation of the C77S mutant relative to WT HiPIP. Ser ligation ultimately results in decreased cluster stability due to increased sensitivity to proton mediated degradation.

Bacterial Proteins↗
PubMed2002

Molybdenum and tungsten in biology.

Molybdenum is the only second-row transition metal that is required by most living organisms, and the few species that do not require molybdenum use tungsten, which lies immediately below molybdenum in the periodic table. Because of their unique chemical versatility and unusually high bioavailability these two transition metals have been incorporated into the active sites of enzymes over the course of evolution. Enzymes that contain molybdenum or tungsten continue to be discovered and several crystal structures have become available recently. This new structural information has been complemented by spectroscopic and kinetic methods, as well as computational approaches. Together, these studies provide an increasingly detailed view of the reaction mechanisms and the correlation between the electronic structure of the active site and catalytic function, one of the fundamental goals in metallobiochemistry.

Aldehyde Oxidoreductases↗
PubMed1999

Freeze-Quench Magnetic Circular Dichroism Spectroscopic Study of the "Very Rapid" Intermediate in Xanthine Oxidase.

Freeze-quench magnetic circular dichroism spectroscopy (MCD) has been used to trap and study the excited-state electronic structure of the Mo(V) active site in a xanthine oxidase intermediate generated with substoichiometric concentrations of the slow substrate 2-hydroxy-6-methylpurine. EPR spectroscopy has shown that the intermediate observed in the MCD experiment is the "very rapid" intermediate, which lies on the main catalytic pathway. The low-energy (< approximately 30 000 cm(-1)) C-term MCD of this intermediate is remarkably similar to that of the model compound LMoO(bdt) (L = hydrotris(3,5-dimethyl-1-pyrazolyl)borate; bdt = 1,2-benzenedithiolate), and the MCD bands have been assigned as dithiolate S(ip) --> Mo d(xy) and S(op) --> Mo d(xz,yz) LMCT transitions. These transitions result from a coordination geometry of the intermediate where the Mo=O bond is oriented cis to the ene-1,2-dithiolate of the pyranopterin. Since X-ray crystallography has indicated that a terminal sulfido ligand is oriented cis to the ene-1,2-dithiolate in oxidized xanthine oxidase related Desulfovibrio gigas aldehyde oxidoreductase, we have suggested that a conformational change occurs upon substrate binding. The substrate-mediated conformational change is extremely significant with respect to electron-transfer regeneration of the active site, as covalent interactions between the redox-active Mo d(xy) orbital and the S(ip) orbitals of the ene-1,2-dithiolate are maximized when the oxo ligand is oriented cis to the dithiolate plane. This underlies the importance of the ene-1,2-dithiolate portion of the pyranopterin in providing an efficient superexchange pathway for electron transfer. The results of this study indicate that electron-transfer regeneration of the active site may be gated by the orientation of the Mo=O bond relative to the ene-1,2-dithiolate chelate. Poor overlap between the Mo d(xy) orbital and the S(ip) orbitals of the dithiolate in the oxidized enzyme geometry may provide a means of preventing one-electron reduction of the active site, resulting in enzyme inhibition with respect to the two-electron oxidation of native substrates.

Journal Article↗