Crystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods

Abstract

The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…Therefore, our current interpretation for the nitrate species is a distorted sixcoordinated site, in which the Mo(V) ion coordinates the four sulfurs of the two pterin cofactors, S c -Cys 140 and one sixth ligand, presumably an oxo group (Scheme 1b). This is in contradiction with the structural data taken for the oxidized forms of Dd NapA [20] and other closely related enzymes [24], which indicated a hydroxyl/ water ligand in the position of the oxo group (Scheme 1a). Additional work is necessary to resolve this discrepancy.…”

“…The rhombic character of this signal excludes Mo(V) ion sites having coordination symmetries such as square pyramidal. On the other hand, on the basis of electron-nuclear double resonance studies of Pp NapAB [30] and the crystal structure of as-prepared Dd NapA [20], it was suggested that the nonsolvent exchangeable hyperfine couplings of the high g (resting) and azide signals in Pp NapAB originate from the protons of the b-methylene carbon of the cysteine coordinated to the oxidized Mo site. Since Dd NapA and Pp NapAB show nitrate and high g EPR signals with hyperfine couplings of similar type and magnitude (Table 1), Cys 140 should be coordinated to this Mo(V) redox state in Dd NapA.…”

“…The 3D X-ray structure of Dd NapA was the first reported for a Nap enzyme [20]. This is a monomeric protein with a funnel-like cavity formed from the surface to the active site (approximately 15 Å ), which is suggested to be the path for nitrate entrance and nitrite exit.…”

“…Rs NapAB [21] and Pp NapAB [22] are heterodimeric proteins, but with their catalytic subunits closely related to Dd NapA. The structures of the catalytic subunits of Dd NapA [20] and Rs NapA [23] are very similar in terms of metal cofactor content, global fold, and domain organization. Although the primary sequences of Dd NapA reveal a low identity (approximately 35%) with Rs NapA and Pp NapA, the residues involved in both Mo-bisMGD cofactor and [4Fe-4S] cluster binding are conserved [24].…”

“…The active site in the oxidized forms of Dd NapA and Rs NapAB is formed by a distorted hexa-coordinated Mo(VI) ion, in which the metal atom is coordinated by four sulfur atoms from two dithioline ligands, one hydroxo/water group, and S c from Cys 140 (in Dd NapA numbering) [20]. In contrast, on the basis of electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) studies, a hepta-coordinated molybdenum site was postulated for Pp NapAB [25], in which an additional oxo group constitutes the main difference from the oxidized active sites of Dd NapA and Rs NapAB [20,23], whereas a negatively charged hepta-coordinated complex was proposed for the reduced form. The structure of the reduced active site in Dd NapA and Rs NapAB has not been reported.…”