D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of Damino acids to the corresponding a-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that ofp-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate a-hydrogen to the flavin during the enzyme reductive half-reaction. The active site of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups of these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalyze similar reactions (oxidation of a-amino acids or a-hydroxy acids), although with opposite stereochemistry.Since the description of D-amino acid oxidase (EC 1.4.3.3; DAAO) activity in mammalian tissues by Krebs in 1935 (1), DAAO has been the subject of a number of biochemical, spectroscopic, and kinetic investigations, becoming the prototype for the oxidase class of the flavin-containing enzymes [for a recent review, see ref. 2]. Its primary structure has been determined and its gene has been cloned (3, 4). Its kinetic and mechanistic properties have been studied in detail by a variety of techniques, while information on the topology of the active site and on its three-dimensional structure have only been derived from chemical modification studies and site-directed mutagenesis of selected residues. Based on these approaches, a catalytic mechanism for DAAO has been proposed, although definitive evidence against alternative mechanisms has not been found (refs. 2 and 5 and references therein).The enzyme catalyzes the oxidation of D-a-amino acids into the corresponding a-ketoacids. The reaction formally proceeds according to the following scheme:E-FADH2 + 02-*E-FAD + H202[2]The reductive half reaction (Eq. 1), in which the noncovalently bound FAD becomes reduced, is followed by the oxidative step in which FAD is reoxidized by molecular oxygen, with the release of hydrogen peroxide (Eq. 2). The imino acid product spontaneously hydrolyzes to the ketoacid in a nonenzymatic process (Eq. 3). DAAO displays a broad substrate specificity, with a preference for D-amin...

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Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of "truncated hemoglobin" N (trHbN), formally acting as NO-dioxygenase, yielding the harmless nitrate ion. X-ray crystal structures have shown that trHbN hosts a two-branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate-limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 micros), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy-trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual-path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification.

The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.