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Theory and Modeling of Biological Systems Theoretical Insight to the Hydroxylamine Oxidoreductase Substrate Binding Site María Laura Fernández, Sara Elizabeth Bari, Darío A. Estrin. Departamento de Química Inorgánica, Analítica y Química Física- INQUIMAE/CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Ciudad Universitaria, Pabellón 2, C1428EHA, Nuñez, Buenos Aires, Argentina. e-mail: bari@qi.fcen.uba.ar Hydroxylamine oxidoreductase from the autotrophic bacteria Nitrosomonas europaea catalyzes the conversion of hydroxylamine to nitrite, with the help of a complicate arrangement of 24 heme groups. It is a homotrimer, and the three subunits are peculiarly linked through three covalent bonds between a C2 of a tyrosine residue of one subunit and a 5-meso carbon atom of c-type heme of another. The linker meso substituted heme group (P460, due to the absorbance of the FeII form); the other seven hemes are c type hemes, optimized for electron transfer.[1, 2] The remarkable complexity of this scenario should be the starting point to explain the efficiency of both the catalytic reaction and energy storage. In the present work, we focused on the influence of the peculiar monomeso pattern of substitution of the P460 binding site in the efficient conversion of hydroxylamine to nitrite from a theoretical perspective, through electronic structure calculations at the density functional theory level (DFT), using the SIESTA code.[3] The nonplanar distortion modes of the P460 have been calculated starting from the atomic resolution crystal structure of the protein, [4] using the normal-coordinate structural decomposition procedure, [5] for the wildtype and for the protein lacking the covalent linkage. Geometry optimizations of the P460 reaction intermediates previously proposed [6] were used to calculate the dissociation energy of different nitrogen containing ligands (NH2OH, NO, HNO), with and without the meso tyrosine residue. The tyrosine residue enhances the binding of the natural substrate hydroxylamine, and benefits the stability of a FeIIINO intermediate, while it shows to behave indifferently in the FeIINO form. Characterization of the non planar heme distortion modes indicated that the meso substituent induces significant ruffling in all the intermediates considered. Nitric oxide binding profile was found to be consistent with that of other ruffled heme models [7] and nitrophorins. [8] References: [1]. Hooper AB and Nason A. J. Biol. Chem. 1965; 240: 4044-57. [2]. Arciero DM and Hooper AB. J. Biol. Chem. 1993; 268: 14645-54. [3]. Soler JM, Artacho E, Gale J, García A, Junquera J, Ordejón P and Sánchez-Portal D. J. Phys: Condens. Matter 2002, 14, 2745. [4]. Igarashi N, Moriyama H, Fuyiwara T, Fukuomori Y and Tanaka N. Nat. Struct. Biol. 1997, 4, 276-84. [5]. Jentzen W, Song, X-Z and Shelnutt, JA. J. Phys. Chem. B 1997, 101, 1684. [6]. Cabail MZ and Pacheco AA. Inorg. Chem. 2003; 42, 270-72. [7]. Linker DP and Rodgers KR. Inorg. Chem. 2005, 44, 1367. [8].Walker FA. J. Inorg. Biochem. 2005, 99, 216. Acknowledgments: UBA, CONICET and ANPCyT for financial support.