Information from e.p.r. spectroscopy on the iron-sulphur centres of the iron-molybdenum protein (aldehyde oxidoreductase) of Desulfovibrio gigas.
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ABSTRACT:
SUBMITTER: Bray RC
PROVIDER: S-EPMC1130529 | biostudies-other | 1991 Dec
REPOSITORIES: biostudies-other
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Project description:The molybdenum iron-sulphur protein originally isolated from Desulfovibrio gigas by Moura, Xavier, Bruschi, Le Gall, Hall & Cammack [(1976) Biochem. Biophys. Res. Commun. 72, 782-789] has been further investigated by e.p.r. spectroscopy of molybdenum(V). The signal obtained on extended reduction of the protein with sodium dithionite has been shown, by studies at 9 and 35 HGz in 1H2O and 2H2O and computer simulations, to have parameters corresponding to those of the Slow signal from the inactive desulpho form of various molybdenum-containing hydroxylases. Another signal obtained on brief reduction of the protein with small amounts of dithionite was shown by e.p.r. difference techniques to be a Rapid type 2 signal, like that from the active form of such enzymes. In confirmation that the protein is a molybdenum-containing hydroxylase, activity measurements revealed that it had aldehyde:2,6-dichlorophenol-indophenol oxidoreductase activity. No such activity towards xanthine or purine was observed. Salicylaldehyde was a particularly good substrate, and treatment of the protein with it also gave rise to the Rapid signal. Molybdenum cofactor liberated from the protein was active in the nit-1 Neurospora crassa nitrate reductase assay. It is concluded that the protein is a form of an aldehyde oxidase or dehydrogenase. From the intensity of the e.p.r. signals and from enzyme activity measurements, 10-30% of the protein in the sample examined appeared to be in the functional form. The evolutionary significance of the protein, which may represent a primitive form of the enzyme rather than a degradation product, is discussed briefly.
| S-EPMC1147922 | biostudies-other
Project description:XOR (xanthine oxidoreductase) purified from human milk was shown to contain 0.04 atom of Mo and 0.09 molecule of molybdopterin/subunit. On the basis of UV/visible and CD spectra, the human enzyme was approx. 30% deficient in iron-sulphur centres. Mo(V) EPR showed the presence of a weak rapid signal corresponding to the enzyme of low xanthine oxidase activity and a slow signal indicating a significant content of desulpho-form. Resulphuration experiments, together with calculations based on enzymic activity and Mo content, led to an estimate of 50-60% desulpho-form. Fe/S EPR showed, in addition to the well-known Fe/S I and Fe/S II species, the presence of a third Fe/S signal, named Fe/S III, which appears to replace partially Fe/S I. Comparison is made with similarly prepared bovine milk XOR, which has approx. 15-fold higher enzymic activity and Mo content. Taken along with evidence of low Mo content in the milk of other mammals, these findings add further support to the idea that XOR protein plays a physiological role in milk (e.g. in secretion) equal in importance to its catalytic function as an enzyme.
| S-EPMC1138957 | biostudies-literature
Project description:1. The mid-point reduction potentials of the various groups in xanthine oxidase from bovine milk were determined by potentiometric titration with dithionite in the presence of dye mediators, removing samples for quantification of the reduced species by e.p.r. (electron-paramagnetic-resonance) spectroscopy. The values obtained for the functional enzyme in pyrophosphate buffer, pH8.2, are: Fe/S centre I, -343 +/- 15mV; Fe/S II, -303 +/- 15mV; FAD/FADH-; -351 +/- 20mV; FADH/FADH2, -236 +/-mV; Mo(VI)/Mo(V) (Rapid), -355 +/- 20mV; Mo(V) (Rapid)/Mo(IV), -355 +/- 20mV. 2. Behaviour of the functional enzyme is essentially ideal in Tris but less so in pyrophosphate. In Tris, the potential for Mo(VI)/Mo(V) (Rapid) is lowered relative to that in pyrophosphate, but the potential for Fe/S II is raised. The influence of buffer on the potentials was investigated by partial-reduction experiments with six other buffers. 3. Conversion of the enzyme with cyanide into the non-functional form, which gives the Slow molybdenum signal, or alkylation of FAD, has little effect on the mid-point potentials of the other centres. The potentials associated with the Slow signal are: Mo(VI)/Mo(V) (Slow), -440 +/- 25mV; Mo(V) (Slow)/Mo(IV), -480 +/- 25 mV. This signal exhibits very sluggish equilibration with the mediator system. 4. The deviations from ideal behaviour are discussed in terms of possible binding of buffer ions or anti-co-operative interactions amongst the redox centres.
| S-EPMC1163874 | biostudies-other
Project description:The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope (99)Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.
| S-EPMC2916431 | biostudies-literature
Project description:Magnetic interaction between molybdenum and one of the iron-sulphur centres in milk xanthine oxidase [Lowe, Lynden-Bell & Bray (1972) Biochem. J. 130, 239-249] was studied further, with particular reference to the newly discovered Mo(V) e.p.r.(electron-paramagnetic-resonance) signal, Resting II [Lowe, Barber, Pawlik & Bray (1976) Biochem. J. 155, 81-85]. E.p.r. measurements at 35GHz near to 4.2K showed that the interaction has the same sign at all molybdenum orientations and is ferromagnetic. The predicted splitting of the e.p.r. signal from the reduced iron-sulphur centre, Fe/S I, was observed, Providing positive identification of this as the other interacting species. Chemical modification of the molybdenum environment in xanthine oxidase can change the size of the interaction severalfold, but interaction always remains approximately isotropic. The interaction in turkey liver xanthine dehydrogenase is indistinguishable from that in the oxidase. However, a bacterial xanthine dehydrogenase with different iron-sulphur centres shows rather larger interaction. Guanidinium chloride disturbs the iron-sulphur centres of the oxidase, and when this occurs there is a parallel and relatively small change in the interaction. Removal of flavin from the molecule, or raising the pH to 12.0, changes the interaction slightly without affecting the chromophores themselves. It is concluded that the Fe/S I centre and the Mo are at least 1.0nm and probably nearer 2.5nm apart, and that the conformation of the protein between them is relatively stable up to pH 12.
| S-EPMC1183819 | biostudies-other
Project description:The potentials of the couples Mo(IV)--(Mo(V) and Mo(V)--Mo(VI) in nitrate reductase from Escherichia coli K12 were measured as + 180 mV and + 220 mV respectively at pH 7.14. The potentials associated with two other e.p.r. signals, believed to be due to iron--sulphur centres, were measured as + 50 mV and + 80 mV.
| S-EPMC1186429 | biostudies-other
Project description:Mononuclear Mo-containing enzymes of the xanthine oxidase (XO) family catalyze the oxidative hydroxylation of aldehydes and heterocyclic compounds. The molybdenum active site shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. The XO family member aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is an exception as presents in its catalytically competent form an equatorial oxo ligand instead of the sulfido ligand. Despite this structural difference, inactive samples of DgAOR can be activated upon incubation with dithionite plus sulfide, a procedure similar to that used for activation of desulfo-XO. The fact that DgAOR does not need a sulfido ligand for catalysis indicates that the process leading to the activation of inactive DgAOR samples is different to that of desulfo-XO. We now report a combined kinetic and X-ray crystallographic study to unveil the enzyme modification responsible for the inactivation and the chemistry that occurs at the Mo site when DgAOR is activated. In contrast to XO, which is activated by resulfuration of the Mo site, DgAOR activation/inactivation is governed by the oxidation state of the dithiolene moiety of the pyranopterin cofactor, which demonstrates the non-innocent behavior of the pyranopterin in enzyme activity. We also showed that DgAOR incubation with dithionite plus sulfide in the presence of dioxygen produces hydrogen peroxide not associated with the enzyme activation. The peroxide molecule coordinates to molybdenum in a η(2) fashion inhibiting the enzyme activity.
| S-EPMC3877041 | biostudies-literature
Project description:Two distinct iron-sulphur centres of the 'HiPIP' (high-potential iron-protein) type are distinguished in both pigeon heart and ox heart mitochondria. These two species, although both are paramagnetic in the oxidized state, exhibit signals which differ in their detailed line shape, field position, and temperature- and power-dependence. They also exhibit different thermodynamic and kinetic behaviour and are located on opposite sides of the mitochondrial coupling membrane. One of these centres corresponds to Centre S-3. The other 'HiPIP'-type centre is removed readily from the mitochondrial membrane and its physiological function is not known.
| S-EPMC1172538 | biostudies-other
Project description:Photosynthesis converts solar energy to chemical energy using chlorophylls (Chls). In a late stage of biosynthesis of Chls, dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR), a nitrogenase-like enzyme, reduces the C17 = C18 double bond of Pchlide and drastically changes the spectral properties suitable for photosynthesis forming the parental chlorin ring for Chl a. We previously proposed that the spatial arrangement of the proton donors determines the stereospecificity of the Pchlide reduction based on the recently resolved structure of the DPOR catalytic component, NB-protein. However, it was not clear how the two-electron and two-proton transfer events are coordinated in the reaction. In this study, we demonstrate that DPOR initiates a single electron transfer reaction from a [4Fe-4S]-cluster (NB-cluster) to Pchlide, generating Pchlide anion radicals followed by a single proton transfer, and then, further electron/proton transfer steps transform the anion radicals into chlorophyllide (Chlide). Thus, DPOR is a unique iron-sulphur enzyme to form substrate radicals followed by sequential proton- and electron-transfer steps with the protein folding very similar to that of nitrogenase. This novel radical-mediated reaction supports the biosynthesis of Chl in a wide variety of photosynthetic organisms.
| S-EPMC4071322 | biostudies-literature
Project description:1. Measurements were made at 12 degrees K of the electron-paramagnetic-resonance (e.p.r.) spectra of submitochondrial particles from Candida utilis cells grown under conditions that alter the amount of the mitochondrial NADH dehydrogenase (EC 1.6.99.3). 2. Iron-limited growth decreases the extent of iron-sulphur e.p.r. signals to undetectable values that are less than 1 percent of those normally found with glycerol-limited growth. 3. Small but significant signals attributable to the NADH dehydrogenase were detected in submitochondrial particles from sulphate-limited cells. 4. Measurements made on submitochondrial particles prepared from these and other phenotypically modified cells lead us to conclude that the presence of low-temperature e.p.r.-detectable iron-sulphur centres attributable to the NADH dehydrogenase are necessary but not sufficient for the coupling of ATP synthesis to the NADH dehydrogenase reaction in the mitochondrial membrane of C. utilis. 6. The amplitude of the g=2.01 signal observed in non-reduced submitochondrial particles is approximately tenfold diminished by iron limitation but not significantly altered by sulphate limitation.
| S-EPMC1165293 | biostudies-other