Quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases.

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.

Purification and properties of alcohol oxidase from Pichia putida.

Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 +/- 4 kDa), and 8-9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422 D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2-3 cysteine residues. The pH optimum was within 8.5-9.0. Specific activity of the enzyme varied from 10 to 50 micromol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20 degrees C and seemed to be stable in phosphate-citrate buffer with 30-50% (w/v) of sucrose. Different forms of 0.1-1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.

[Purification and properties of membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)].

Membrane fraction of Methylococcus capsulatus (strain M) were treated with [14C]acetylene, an affinity label binding to the active center of membrane-bound methane monooxygenase (MMO). High-purity particulate form of methane hydroxylase (pMH) was obtained by ion exchange and hydrophobic chromatography. According to SDS-PAGE data, the enzyme contained three polypeptides with molecular weights of 47 (alpha), 27 (beta), and 25 (gamma) kDa in the ratio 1:1:1. The radiolabel was contained in the beta-subunit of pMH. The protein contained 1 or 2 atoms of nonheme iron and 2-4 atoms of copper per a minimum molecular weight of 99 kDa. This protein did not oxidize methane or propylene in the presence of NADH but was able to oxidize low quantities of methane in the presence of duroquinol. It was established that methanol dehydrogenase (MD) and NADH oxidoreductase (NADH-OR) are peripheral membrane proteins. Possible causes of low activity of high-purity methane hydroxylase are discussed.

[The binuclear iron site of the membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)].

The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: 47 (alpha), 27 (beta), and 25 kDa (gamma). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4 degrees C exhibited, along with the copper signal of type I with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00. After incubation at -30 degrees C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at -30 degrees C. It was established that this intermediate is a high-spin dimer [Fe(IlI)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(lII)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the beta-subunit and cannot be detected by ESR. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M.

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.

Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments.

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

Electron microscopy of the Mo-Fe-protein from Azotobacter vinelandii nitrogenase.

The quaternary structure of the Mo-Fe-protein from Azotobacter vinelandii has been studied by electron microscopy. A model of the molecule of the Mo-Fe-protein has been proposed: two alpha subunits are displaced relative to two beta subunits along a twofold axis, so the molecule can be characterized by the point-group pseudosymmetry 222. Computer averaging of the images showed that one of the projections of the molecule could be characterized by twofold rotational symmetry. Micrographs of nitrogenase recombined complex (Mo-Fe-protein + Fe-protein) have been obtained. They showed particles close in size and form to the Mo-Fe-protein molecule. Therefore, it has been proposed that the Fe-protein could be situated in the central cavity of Mo-Fe-protein.

[Role of CO-binding cytochrome c in enzymatic oxidation of methane by the bacterium Methylococcus capsulatus]. / Rol' tsitokhroma c, sviazyvaiushchego CO, v fermentativnom okislenii metana bakteriei Methylococcus capsulatus.

The cytochrome c spectrally related to cco cytochromes has been isolated and purified from the methane-oxidizing bacterium Methylococcus capsulatus. The cytochrome binds CO but does not bind other substrates of methane monooxygenase, does not activate the methane monooxygenase reaction and is not a component of methane monooxygenase. In the methanol dehydrogenase enzymatic system cytochrome cco functions as electron acceptor. A possible role of cytochrome cco as electron carrier intermediate in the sequence of the dehydrogenase and oxidase enzymatic systems of M. capsulatus is discussed.

[Study of the amino acid residue topography of the adenosine triphosphatase center of (Ca--Mg)-dependent sarcoplasmic reticulum adenosine triphosphatase by kinetic and spectral methods]. / Issledovanie topografii aminokislotnykh ostatokov adenozintrifosfataznogo tsentra (Ca--Mg)-zavisimoi adenozintrifosfatazy sarkoplazmaticheskogo retikuluma kineticheskimi i spektral'nymi metodami.

The topography of HS- and NH2-groups and tryptophane residues in ATPase centre of (Ca--Mg)-ATPase on sarcoplasmic reticulum (SR) was investigated by kinetics, electron spectroscopy and spectrofluorimetry method. Both o-phthalaldehyde interacting with lysine or arginine residue or with end amino acid and fluorescein dimercuric acetate interaction with cysteine residue of HS-groups make (Ca--Mg)-ATPase both in SR and the pure enzyme completely inactive at molar ratio enzyme: inhibitor equal to 1 : 1. A 500 molar ATP surplus reduces drastically the enzyme inactivation rate by both inhibitors. The data supplied by the spectrofluorimetry and the induction-resonance theory were used to calculate the distances between nearest tryptophane residues and chromophore (o-FTC) generated by o-phthalaldehyde interaction with NH2-group the protein amino acid residue (17 A) and o-FTC and fluorescein dimercuric acetate (19 A) attached to enzyme HS-group. Because o-FTC is inside the protein pocket it is not accessible to J- ions up to 2.5 M KJ. However some tryptophane resudies and fluorescein dimercuric acetate attached to HS-group are near to the macromolecule surface. Lysine (or arginine residues) or end amino acid NH2-group and cysteine residues HS-group, and some tryptophane residues are at ATPase centre of (Ca--Mg)-ATPase from sarcoplasmic reticulum. Possible topography of the centre is discussed.

[Estimation of the distance between the iron-sulfur cluster of Fe-protein and the nearest iron-sulfur cluster of Mo-Fe-protein of nitrogenase on the basis of the inductive-resonance theory of energy transfer]. / Otsenka rasstoianiia mezhdu zhelezo-sernym klasterom FE-belka i blizhaishim zhelezo-sernym klasterom Mo-Fe-belka nitrogenazy na osnovanii teorii induktivno-rezonansnogo perenosa énergii.

The distance between fluorescein mercuric acetate (FMA), attached to the HS-group of Fe- and Mo-Fe-protein, and the nearest iron-sulphur cluster (ISC) was determined. For Fe-protein the distance was 18--20 A and for Mo-Fe-protein 12--14 A. The distance between Fe-protein FMA and the nearest Mo-protein ISC determined by complementation of the labelled Fe-protein and native Mo-Fe-protein was 14--16 A. The distance between MO-OFe-protein ISC and complement Fe-protein ISC was 18--20 A. A te-protein ISC permitted to suppose that the electron was transfered from Fe-protein ISC to Mo-Fe-protein ISC by the contact of the ISC or with the help of ATP molecule.

[Method for assaying methane monooxygenase activity by measuring methane uptake in the reaction vessel]. / Opredelenie aktivnosti metanmonooksigenazy po ubyli metana b reaktsionnoi srede.

The reaction vessel has been designed to measure methane monooxygenase activity. An elastic membrane has been built into one of the walls of the vessel to take liquid samples, avoiding formation of the gaseous phase in the reaction volume. The methane content in the samples is measured in a gas-liquid chromatograph with a flame ionization detector in two ways: 1. by direct measurement of methane in the liquid sample, and 2. by measurement of methane in the gaseous phase after methane diffusion from the liquid sample into the gaseous space of another vessel. The method is simple, sensitive (with a lower limit of 0.1 nMole CH4), and well reproducible. This method permits measurement of the oxidation kinetics of methane and other gaseous hydrocarbons both by intact cells and cell-free preparations of methane oxidizing bacteria.

Structure and mechanism of catalytic action of active sites of nitrogenase.

A review of the data on the macromolecular structure of nitrogenase and its individual fragments, the electronic structure of iron- and molybdenum-containing components of the active site, and the functional groups of the ATPase site of the enzyme is given. Reactions of N2 reduction, ATP hydrolysis, and H2 evolution, inhibitory processes, and electron transport reactions catalyzed by the enzyme are analyzed within the framework of a general kinetic model. The results of an investigation of the location of the iron-containing cluster system of electron transport, the ATPase site, and the N2-binding and reducing site on the nitrogenase macro-molecule with the aid of a new complex approach including methods of spin, luminescent, and electron-dense labeling are described. On the basis of a number of physicochemical and kinetic data a model of the structure and mechanism of action of the active site of nitrogenase is proposed, which assumes four-step electron transfer from an external reducing agent along the chain of ferredoxin-like iron-containing clusters of the enzyme and an increase in the reducing potential of the iron clusters through the energy of ATP hydrolysis and four-electron reduction in a binuclear molybdenum-containing complex.

[Isotopic effect in enzymatic oxidation of methane]. / Izotopnyi effekt v fermentativnom okislenii metana

The isotopic effect during oxidation of methane and deuteromethane by a suspension of Methylomonas rubrum cells, for which methane is the only source of carbon, was observed. The rate of CH4 oxidation is 12.5 times higher than that of CH4 oxidation. It is demonstrated that CD4 is a competitive inhibitor of CH4 oxidation. The results obtained suggest that the disruption of the C-H bond is the limiting step of enzymatic oxidation of methane.