EXAFS nitrate reductase

Study by X-ray absorption spectroscopy of the extended X-ray absorption fine structure (EXAFS) has provided estimates of both the nature and the number of the nearest neighboring atoms around the Mo. The EXAFS spectra of xanthine dehydrogenase and of nitrate reductase from Chlorella confirmed the... 

Nitrate reductase from Chlorella, an assimilatory enzyme, is a homotetramer of molecular weight 360 000 and contains one each of Mo, heme and FAD per subunit. The nitrate reductase from E. coli is a dissimilatory enzyme. EXAFS data are available on the molybdenum sites in both enzymes (Table 24).1050 The environment of the molybdenum in the assimilatory enzyme is similar to that found for sulfite oxidase, with at least two sulfur ligands near the molybdenum and a shuttle between monoxo and dioxo forms with redox change in the enzyme. This allows a similar mechanism to be put forward for the assimilatory nitrate reductase,1051 shown in equation (57), where an oxo group is transferred from nitrate to MoIV with production of nitrite and MoVI. 

Both assimilatory and dissimilatory nitrate reductases are molybdoenzymes, which bind nitrate at the molybdenum. EXAFS studies1050 have shown that there are structural differences between the assimilatory nitrate reductase from Chlorella vulgaris and the dissimilatory enzyme from E. coli. The Chlorella enzyme strongly resembles sulfite oxidase1050,1053 and shuttles between mon-and di-oxo forms, suggesting an oxo-transfer mechanism for reduction of nitrate. This does not appear to be the case for the E. coli enzyme, for which an oxo-transfer mechanism seems to be unlikely. The E. coli enzyme probably involves an electron transfer and protonation mechanism for the reduction of nitrate.1056 It is noteworthy that the EXAFS study on the E. coli nitrate reductase showed a long-distance interaction with what could be an electron-transfer subunit. 

Protein sequence homology suggests that sulfite oxidase and assimilatory nitrate reductase are members of the same molybdenum enzyme subfamily [31]. Consistent with this classification, the cofactors of sulfite oxidase and assimilatory nitrate reductase differ significantly from those in dmso reductase, aldehyde oxido-reductase, xanthine oxidase (see Section IV.E.), and even respiratory nitrate reductase (Section IV.D). The EXAFS of both sulfite oxidase [132-136] and assimilatory nitrate reductase [131,137,138] and x-ray studies of sulfite oxidase (chicken liver) [116] confirm that the molybdenum center is coordinated by two sulfur atoms from a single MPT ligand and by the sulfur atom of a cysteine side chain. The Movl state is bis(oxido) coordinated (Figure 14). 

Figure 13 The coordination geometry around molybdenum as suggested from EXAFS and genetic studies of respiratory nitrate reductase (E. coli) (a) oxidized form (b) reduced form [131],...

As with xanthine oxidase, the sulfido ligand of the active form of aldehyde oxidoreductase is readily replaced by an oxido ligand to yield a cofactor with a structure that resembles that of oxidized sulfite oxidase and assimilatoiy nitrate reductase. Both x-ray and EXAFS data are available for the bis(oxido) form, and, with the exception of the oxido replaced sulfido ligand, few changes are obvious in the overall structure of the oxidized form of the desulfo cofactor. Upon reduction of the enzyme the oxido ligand is presumably reduced to hydroxido, an observation that is supported by EPR data for the Mov state. 

A striking feature of these EXAFS studies is the observation that a substantial proportion of the molecules of E. coli nitrate reductase—in the reduced form at pH 6.8 and the oxidized and reduced forms at pH 10.2—appear to lack oxo groups. Such systems appear to be unique among the oxo molybdoenzymes. 

The nature of the molybdenum centers of several dissimilatory nitrate reductases has been investigated by K-edge EXAFS and by EPR spectroscopy " " " and the reduction potentials of the several redox-active centers in the E. coli nitrate reductase have also been determined by following the intensity of the low pH EPR signal as a function of the poised system potential. " The redox potentials of the Mo(VI/V) and Mo(V/IV) couples were determined as... 

However, some of these structures show unusual features and a crowded active site. EXAFS studies of Rhodobacter capsulatus DM SO reductase show the expected four Mo-S ligands and one Mo-O bond arising from a serine residue plus one Mo=0 in the reduced Mo (IV) state of the active site. This oxo ligand is removed upon oxidation of the metal ion to Mo(VI). In the oxidized structme, an aquo ligand is postulated to coordinate the molybdenum ion (Fig. 11.17c) [126-128]. An equivalent restdt has been reported for BSO that catalyzes the reduction of D-biotin D-sulfoxide to D-biotin [129]. A similar overall catalytic mechanism is expected for nitrate reductases (Fig. 11.18), which catalyze the following reaction ... 

---