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.

The molybdenum [iron-sulfur] protein, first isolated from Desulfovibrio gigas by Moura et al. [Moura, J. J. G., Xavier, A. V., Bruschi, M., Le Gall, J., Hall, D. O., & Cammack, R. (1976) Biochem. Biophys. Res. Commun. 72, 782-789], was later shown to mediate the electronic flow from salicylaldehyde to a suitable electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) [Turner, N., Barata, B., Bray, R. C., Deistung, J., LeGall, J., & Moura, J. J. G. (1987) Biochem. J. 243, 755-761]. The DCPIP-dependent aldehyde oxidoreductase activity was studied in detail using a wide range of aldehydes and analogues. Steady-state kinetic analysis (KM and Vmax) was performed for acetaldehyde, propionaldehyde, benzaldehyde, and salicylaldehyde in excess DCPIP concentration, and a simple Michaelis-Menten model was shown to be applicable as a first kinetic approach. Xanthine, purine, allopurinol, and N1-methylnicotinamide (NMN) could not be utilized as enzyme substrates. DCPIP and ferricyanide were shown to be capable of cycling the electronic flow, whereas other cation and anion dyes [O2 and NAD(P)+] were not active in this process. The enzyme showed an optimal pH activity profile around 7.8. This molybdenum hydroxylase was shown to be part of an electron-transfer chain comprising four different soluble proteins from D. gigas, with a total of 11 discrete redox centers, which is capable of linking the oxidation of aldehydes to the reduction of protons.

The Desulfovibrio gigas aldehyde oxido-reductase contains molybdenum and iron-sulfur clusters. Mossbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxydized enzyme, the clusters are diamagnetic and exhbit a single quadrupole doublet with parameters ( A EQ = 0.62 0.01 mm/s) typical for the [2Fe-2SI2+ state. Mossbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2Sj1 Molybdenum hydroxylases are an important group of proteins within the molybdenum-containing enzymes [l -31. In t h s class of enzymes, the molybdenum is found in a pterin cofactor (termed Mo-co), which contains no other transition metal besides molybdenum [4]. A different situation is found in the case of another group of molybdenum-containing enzymes, namely nitrogenase, where the cofactor is a spin-coupled cluster of molybdenum and iron atoms (termed the FeMo-co) [5,6]. Another difference, with respect to the redox centers of these two classes of enzymes, is that nitrogenase contains four putative [4Fe-4S] clusters (termed the P-clusters) [7, 81, while the molybdenum hydroxylases, such as xanthine oxidase, xanthine dehydrogenase and aldehyde oxidase, contain two spectroscopically distinguishable [2Fe-2S] clusters (termed Fe/S I and Fe/S 11) [2, 91. A molybdenum [iron-sulfur] protein was isolated from Desulfovibrio gigas by Mourd et al. [lo, 111. Previous studies have focused on the molybdenum site. Extended X-ray-absorption fine structure (EXAFS) spectra of this protein indicated a molybdenum environment similar to that in the desulfo form of xanthine oxidase [12]. Detailed investigations using the EPR technique revealed that extended reduction of the protein by dithionite yielded a molybdenum signal similar to that of the inactive desulfo form of various molybdenumcontaining hydroxylases, while brief reduction of the protein generated an additional molybdenum signal similar to that of the active form of those enzymes [33]. Activity measurements In this manuscript, we present a re-determination of the metal content of this D . gigas molybdenum [iron-sulfur] protein (termed aldehyde oxido-reductase) and report a detailed Mossbauer characterization of its iron-sulfur clusters. The data unambiguously demonstrate that this protein contains four iron atoms arranged in two spectroscopically distinguishable [2Fe-2S] clusters. A Mossbauer study of the protein reacted with benzaldehyde shows partial reduction of the [2Fe-2S] clusters, indicating the involvement of the clusters in the process of substrate oxidation. MATERIALS AND METHODSProtein purification and sample preparation "Fe enrichment of the bacterial mass was accomplished as reported in [14]. The D. gigus aldehyde oxido-reductase was isolated essentially as described in [Ill, using an additional H...

E.p.r. spectra of reduced iron-sulphur centres of the aldehyde oxidoreductase (iron-molybdenum protein) of Desulfovibrio gigas were recorded at X-band and Q-band frequencies and simulated. Results are consistent with the view that only two types of [2Fe-2S] clusters are present, as in eukaryotic molybdenum-containing hydroxylases. The data indicate the Fe/SI centre to be very similar, and the Fe/SII centre somewhat similar, to these centres in the eukaryotic enzymes.

The Desulfovibrio gigas aldehyde oxidoreductase contains molybdenum bound to a pterin cofactor and [2Fe-2S] centers. The enzyme was characterized by SDSPAGE, gel-filtration and analytical ultracentrifugation experiments. It was crystallized at !"C, pH 7.2, using isopropanol and MgC1, as precipitants. The crystals diffract beyond 0.3-nm (3.0-A) resolution and belong to space group P6,22 or its enantiomorph, with cell dimensions u = b = 14.45 nm and c = 16.32 nm. There is one subunit/asymmetric unit which gives a packing density of 2.5 X nm'/Da (2.5 A3/Da), consistent with the experimental crystal density, p = 1.14 g/cm3. One dimer (approximately 2 X 100 kDa) is located on a crystallographic twofold axis.The detailed knowledge of the structure and the catalytic mechanism of molybdenum-containing enzymes has considerably increased during the last decade [I -61 and, in particular, the biochemistry of aldehyde oxidoreductases has been extensively studied [4, 51. Molybdenum is a relevant transition metal in biological systems. Two groups of molybdo-proteins have been described. In the first group Mo is associated with iron in a complex cluster type structure (FeMocofactor) in the nitrogenase enzyme. Recently, a structural model of the FeMoco was proposed based on crystallographic analysis, consisting of [4Fe,3S] and [lMo,3Fe,3S] clusters bridged by three nonprotein ligands [7, 81. In the second group Mo is contained in an organic structural component (pterin), designated as Mocofactor (molybdopterin), generally associated with ironsulfur centers and/or a flavin or heme center, as in the case of molybdenum oxotransferases [6].From sulfate-reducing bacteria (strict anaerobes) enzymes related to the second group have been isolated and characterized [9-171. They represent unique situations for the presence of such a group of enzymes in the prokaryotic world, since most often these proteins are studied in eukaryotes (e.g. plants, insects and higher animals).The molybdenum iron-sulfur protein (MOP) isolated from D. gigas has analogies with the molybdenum hydrox- An important advance on these studies was the possibility to isolate the enzyme from 57Fe-enriched media with obvious interest for an iron-sulfur-center site labelling, enhanced sensitivity of the Mossbauer studies (an advantage with respect to mammalian systems) and the possibility of a direct measurement of substrate binding [14]. Previously described molybdenum (V) resting-type and slow-type EPR signals and the extended-X-ray-absorption fine-structure (EXAFS) spectrum of the molybdenum center indicated close similarities to desulfo-xanthine oxidase (inactive) [lo-121. In addition, it was later demonstrated for MOP, a new Mo(V) EPR signal (rapid type 2) centered at EPR average g-value (gav) of 1.9742, similar to those observed for xanthine and aldehyde oxidases [12]. These rapid EPR signals have been shown, in these proteins, to be physiologically significant, as they develop within the enzymeturnover time scale. A molybdenum cofactor, liberated from the protei...