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At least 1,243 records · Page 69Linked to original sources
PubMed2005

MauG-dependent in vitro biosynthesis of tryptophan tryptophylquinone in methylamine dehydrogenase.

Tryptophan tryptophylquinone (TTQ) is the prosthetic group of methylamine dehydrogenase (MADH) and is synthesized through post-translational modification of two endogenous tryptophan residues. This modification involves two oxygenation reactions and one cross-linking reaction. It is clearly shown that the incorporation of the second oxygen into betaTrp57 and the covalent cross-linking of betaTrp57 to betaTrp108 are MauG-dependent processes. These reaction steps are severely compromised in vivo when mauG is mutated or deleted. These steps may then be catalyzed in vitro upon addition of MauG to the isolated biosynthetic intermediates. These results also show that TTQ formation is linked to proper assembly of subunits during MADH biosynthesis. Last, these results demonstrate a novel function for the c-type heme protein, MauG, which is consistent with its atypical physical properties. These results are the first description of an enzyme-mediated biosynthesis of a protein-derived cofactor in vitro.

Hemeproteins↗
PubMed1997

Haem-ligand switching during catalysis in crystals of a nitrogen-cycle enzyme.

Cytochrome cd1 nitrite reductase catalyses the conversion of nitrite to nitric oxide in the nitrogen cycle. The crystal structure of the oxidized enzyme shows that the d1 haem iron of the active site is ligated by His/Tyr side chains, and the c haem iron is ligated by a His/His ligand pair. Here we show that both haems undergo re-ligation during catalysis. Upon reduction, the tyrosine ligand of the d1 haem is released to allow substrate binding. Concomitantly, a refolding of the cytochrome c domain takes place, resulting in an unexpected change of the c haem iron coordination from His 17/His 69 to Met106/His69. This step is similar to the last steps in the folding of cytochrome c. The changes must affect the redox potential of the haems, and suggest a mechanism by which internal electron transfer is regulated. Structures of reaction intermediates show how nitric oxide is formed and expelled from the active-site iron, as well as how both haems return to their starting coordination. These results show how redox energy can be switched into conformational energy within a haem protein.

Catalysis↗
PubMed2002

Reduction of cytochrome c oxidase by a second electron leads to proton translocation.

Cytochrome c oxidase, the terminal enzyme of cellular respiration in mitochondria and many bacteria, reduces O(2) to water. This four-electron reduction process is coupled to translocation (pumping) of four protons across the mitochondrial or bacterial membrane; however, proton pumping is poorly understood. Proton pumping was thought to be linked exclusively to the oxidative phase, that is, to the transfer of the third and fourth electron. Upon re-evaluation of these data, however, this proposal has been questioned, and a transport mechanism including proton pumping in the reductive phase--that is, during the transfer of the first two electrons--was suggested. Subsequently, additional studies reported that proton pumping during the reductive phase can occur, but only when it is immediately preceded by an oxidative phase. To help clarify the issue we have measured the generation of the electric potential across the membrane, starting from a defined one-electron reduced state. Here we show that a second electron transfer into the enzyme leads to charge translocation corresponding to pumping of one proton without necessity for a preceding turnover.

Biological Transport↗
PubMed2006

Proton-coupled electron transfer drives the proton pump of cytochrome c oxidase.

Electron transfer in cell respiration is coupled to proton translocation across mitochondrial and bacterial membranes, which is a primary event of biological energy transduction. The resulting electrochemical proton gradient is used to power energy-requiring reactions, such as ATP synthesis. Cytochrome c oxidase is a key component of the respiratory chain, which harnesses dioxygen as a sink for electrons and links O2 reduction to proton pumping. Electrons from cytochrome c are transferred sequentially to the O2 reduction site of cytochrome c oxidase via two other metal centres, Cu(A) and haem a, and this is coupled to vectorial proton transfer across the membrane by a hitherto unknown mechanism. On the basis of the kinetics of proton uptake and release on the two aqueous sides of the membrane, it was recently suggested that proton pumping by cytochrome c oxidase is not mechanistically coupled to internal electron transfer. Here we have monitored translocation of electrical charge equivalents as well as electron transfer within cytochrome c oxidase in real time. The results show that electron transfer from haem a to the O2 reduction site initiates the proton pump mechanism by being kinetically linked to an internal vectorial proton transfer. This reaction drives the proton pump and occurs before relaxation steps in which protons are taken up from the aqueous space on one side of the membrane and released on the other.

Adenosine Triphosphate↗
PubMed2000

Using direct electrochemistry to probe rate limiting events during nitrate reductase turnover.

Protein film voltammetry of NarGH catalysing nitrate reduction under steady state conditions provides information on events occurring within the enzyme during the catalytic cycle. In this discussion we have focused on exploring the ability of two simple catalytic schemes to reproduce the voltammetric response of NarGH; electron transfer to the enzyme's active site being described either by interfacial electron exchange (Scheme 1) or intramolecular electron delivery via the operation of an electron relay centre (Scheme 2). When the two electron reduced, catalytically competent active site of the enzyme is generated from the oxidised form in 'rapid', non-rate limiting steps of the catalytic cycle, the voltammetric behaviour of NarGH cannot be reproduced. Rather under all the conditions investigated, one electron reduction of the active site from a semi-reduced to a fully-reduced state is found to be crucial to progression through the enzyme's catalytic cycle. The catalytically relevant semi- and fully-reduced oxidation states of the NarGH active site are most likely to correspond to the Mo(V) and Mo(IV) states of the Mo(MGD)2 centre, respectively, although it is not possible to rule out the possibility that they correspond to molybdopterin based oxidation states as observed in other enzymes. We suggest that the rate of either conformational rearrangement within the semi-reduced active site or intramolecular electron delivery to the active site constitutes a defining feature in the catalytic cycle of NarGH and results in the napp approximately 1 appearance of the catalytic waveform.

Electrochemistry↗
PubMed1965

THE UTILIZATION OF GLYCOLLATE BY MICROCOCCUS DENITRIFICANS: THE BETA-HYDROXYASPARTATE PATHWAY.

1. Micrococcus denitrificans utilized glycollate as sole carbon source for aerobic growth. Glyoxylate was utilized less well, and though glycine alone did not support growth it enhanced growth on glyoxylate. 2. During growth on glycollate, (14)C was incorporated from [2-(14)C]glycollate into glycine and thence into aspartate, malate and glutamate. No phosphoglycerate was labelled at the earliest times. 3. Glyoxylate was the first product of glycollate utilization, and glycollate oxidase was inducibly formed on transfer of the organism to glycollate-containing media. 4. Extracts of glycollate-grown M. denitrificans contained negligible glyoxylate-carboligase activity and only low tartronate semialdehyde-reductase activity. 5. erythro-beta-Hydroxyaspartate is a key intermediate in glyoxylate utilization by this organism. Enzymes catalysing (a) the synthesis of erythro-beta-hydroxyaspartate from glyoxylate and glycine, and (b) the conversion of erythro-beta-hydroxyaspartate into oxaloacetate, were inducibly formed during growth on glycollate and on other substrates yielding glyoxylate. Methods for the assay of these enzymes were developed. 6. It is concluded that in M. denitrificans the biosynthesis of cell materials from glycollate is accomplished by the ;beta-hydroxyaspartate pathway', a novel metabolic route that may also perform a catabolic role in glyoxylate oxidation.

Acetates↗
PubMed1973

Aminolaevulinate synthetase of Micrococcus denitrificans. Purification and properties of the enzyme, and the effect of growth conditions on the enzyme activity in cells.

1. 5-Aminolaevulinate synthetase was detected in extracts of the non-photosynthetic bacterium Micrococcus denitrificans. 2. Activity is high in cells grown anaerobically in a defined nitrate medium, but is low in cells grown in an iron-deficient medium, and in cells grown aerobically. 3. Aminolaevulinate synthetase was purified extensively; it has a molecular weight of about 68000; apparent K(m) values for glycine, succinyl-CoA and pyridoxal phosphate are 12mm, 10mum and 11mum respectively; 2mum-haemin and 14mum-protoporphyrin inhibit by 50%. 4. The low activity of aminolaevulinate synthetase in iron-deficient cells increases on adding iron salts to cells only under conditions where protein synthesis can occur. 5. In defined nitrate medium with a high Ca(2+) concentration anaerobic growth yield is higher, but aminolaevulinate synthetase activity is lower than in cells grown in the medium with a low Ca(2+) concentration. In medium made from AnalaR constituents, growth yield is low and aminolaevulinate synthetase activity is high even in the presence of high concentrations of Ca(2+); on adding Cu(2+) (0.1mum) to the medium growth yield and aminolaevulinate synthetase activity become the same as in non-AnalaR medium. 6. Cells incubated under conditions where protein synthesis does not occur but where electron transport does, lose their aminolaevulinate synthetase activity rapidly. 7. The activities of aminolaevulinate dehydratase and succinic thiokinase do not change under any of the conditions of growth examined. 8. The possible mechanisms controlling aminolaevulinate synthetase activity and the role of this enzyme in controlling the synthesis of haem in this organism are discussed.

5-Aminolevulinate Synthetase↗
PubMed1975

The identification and biosynthesis of siderochromes formed by Micrococcus denitrificans.

1. Micrococcus denitrificans excretes three catechol-containing compounds, which can bind iron, when grown aerobically and anaerobically in media deficient in iron, and anaerobically in medium with a high concentration of Ca2+. 2. One of these compounds was identified as 2,3-dihydroxybenzoic acid (compound I), and the other two were tentatively identified as N1N8-bis-(2,3-dihydroxybenzoyl)spermidine (compound II) and 2-hydroxybenzoyl-N-L-threonyl-N4[N1N8-bis-(2,3-dihydroxybenzoyl)]spermidine (compound III). 3. The equimolar ferric complex of compound III was prepared; compound III also forms complexes with Al3+, Cr3+ and Co2+ ions. 4. Cell-free extracts from iron-deficient organisms catalyse the formation of compound II from 2,3-dihydroxybenzoic acid and spermidine, and of compound III from compound II, L-threonine and 2-hydroxybenzoic acid; both reactions require ATP and dithiothreitol, and Mg2+ stimulates activity. The enzyme system catalysing the formation of compound II has optimum activity at pH 8.8 Fe2+ (35muM), Fe3+ (35muM) and Al3+ (65muM) inhibit the reaction by 50 percent. The enzyme system forming compound III has optimum activity at pH 8.6. Fe2+ (110 muM), Fe3+ (110 muM) and Al3+ (135 muM) inhibit the reaction by 50 percent. 5. At least two proteins are required for the formation of compound II, and another two proteins for its conversion into compound III. 6. The changes in the activities of these two systems were followed after cultures became deficient in iron. 7. Ferrous 1,10-phenanthroline is formed when a cell-free extract from iron-deficient cells is incubated with the ferric complex of compound III, succinate, NADH and 1,10-phenanthroline under N2.

Adenosine Triphosphate↗
PubMed1975

The autotrophic growth of Micrococcus denitrificans on Methanol.

Ribulose bisphosphate carboxylase is present at a high specific activity in extracts of methanol-grown Microccus denitrificans. Enzymic and physiological evidence indicates that, during growth on methanol, the ribulose bisphosphate cycle is the route of carbon assimilation.

Carbon Dioxide↗
PubMed1993

A method for extracting rate constants from initial rates of stopped-flow kinetic data: application to a physiological electron-transfer reaction.

The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.

Bacterial Proteins↗
PubMed1995

Mechanism of reaction of allylamine with the quinoprotein methylamine dehydrogenase.

Allylamine did not serve as an efficient substrate for methylamine dehydrogenase (EC 1.4.99.3) in a steady-state assay of activity and appeared to act as a competitive inhibitor of methylamine oxidation by methylamine dehydrogenase. Transient kinetic studies, however, revealed that allylamine rapidly reduced the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase. The rate of TTQ reduction by allylamine was 322 s-1, slightly faster than the rate of reduction by methylamine. These data were explained by a kinetic mechanism in which allylamine and methylamine are alternative substrates for methylamine dehydrogenase. The apparent competitive inhibition by allylamine is due to a very slow rate of release of the aldehyde product, 0.28 s-1, relative to a rate of 18.6 s-1 for the release of the aldehyde product of methylamine oxidation. A reaction mechanism is proposed for the oxidative deamination of allylamine by methylamine dehydrogenase. This mechanism is discussed in relation to the reaction mechanisms of topa-bearing quinoprotein amine oxidases, the flavoprotein monoamine oxidase and the mammalian semicarbazide-sensitive amine oxidase.

Allylamine↗
PubMed1995

The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha.

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.

Amino Acid Sequence↗