CRYSTAL STRUCTURE OF A PERIPLASMIC NITRATE REDUCTASE (NAP) FROM DESULFOVIBRIO DESULFURICANS ATCC 27774 AT 1.8 A RESOLUTION BY MAD: A MOLYBDOPTERIN ENZYME WITH A SINGLE FE₄S₄ CLUSTER

Joao M. Dias¹, Manuel Than², Robert Huber², Gleb Bourenkov³, Hans Bartunik³, Sergey Bursakov⁴, Isabel Moura⁴, Jorge Caldeira⁴, José J.G. Moura⁴, Maria J. Romao¹

¹Instituto Superior Técnico, Departamento de Enga Química, Av. Rovisco Pais, 1096- Lisboa Codex and ITQB, Apt. 127, 2780- Oeiras, Portugal
²Max-Planck-Institut für Biochemie, am Klopferspitz 18a, D-82152 Martinsried, Germany.
³Max-Planck Research Unit for Structural Molecular Biology, MPG-ASMB c/o DESY, 22603 Hamburg, Germany
⁴Departamento de Química, Centro de Química Fina e Biotecnologia, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal

Periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans (D. d.) ATCC 27774, catalyses the reduction of nitrate to nitrite and is induced under cell adaptation to nitrate respiration [Moura et al., 1997].

NAP is a molybdenum containing enzyme with two molybdopterin guanine dinucleotide cofactors (MGD) and an Fe₄S₄ cluster in a single polypeptide of 723 amino acid residues. This enzyme was crystallised in space group P3₁21, with unit cell dimensions of a=b=106.14 A and c=134.84 A and these crystals diffract to beyond 1.8 A with synchrotron radiation.

The three-dimensional structure of the oxidised form of the enzyme was solved by the MAD method, as single phasing information (at the iron absorption edge), and refined at 1.8 A, to a crystallographic R-factor of 21.9%, and R-free of 27.6%. In the refined model, the root-mean-square deviation from ideality is 0.01 A for bond distances and 2.0 ° for bond angles.

The structure of NAP is divided in four domains, showing an a/b type folding. All four domains are involved to variable extents in cofactor binding and, while the Fe₄S₄ center is located at the periphery of the molecule in domain I, the bis-MGD molybdenum cofactor is extended across the interior of the molecule and interacts with residues from all four domains

The molybdenum atom is located at the bottom of a deep crevice, ca. 15 A away from the nearest surface and only 12 A distant from the closest Fe atom of the [4Fe-4S] cluster. In analogy to what is observed in the other molybdopterin-containing enzymes (molybdenum-iron protein aldehyde oxido-reductase, MOP [Romao et al., 1995], and formate dehydrogenase [Boyington et al., 1997]) there is a facile electron transfer pathway connecting the molybdenum and iron centers. A reaction mechanism will be proposed.

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