Structural and Kinetic Evidence for an Ordered Mechanism of Copper Nitrite Reductase Richard W. Strange 1 , Loretta M. Murphy 1 , Fraser E. Dodd 1 Zelda H. L. Abraham 2 , Robert R. Eady 2 , Barry E. Smith 2 and S. Samar Hasnain 1 * 1 CCLRC Daresbury Laboratory Warrington, Cheshire WA4 4AD, UK 2 Nitrogen Fixation Laboratory John Innes Centre, Norwich NR4 7HU, UK The crystallographic structures of several copper-containing nitrite reductases are now available. Despite this wealth of structural data, no de®nitive information is available as to whether the reaction proceeds by an ordered mechanism where nitrite binds to the oxidised type 2 site, fol- lowed by an internal electron transfer from the type 1 Cu, or whether binding occurs to the reduced type 2 Cu centre, or a random mechanism operates. We present here the ®rst structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absorption spectroscopy. The reduced type 2 Cu site EXAFS shows striking similarity to the EXAFS data for reduced bovine superoxide dismutase (Cu 2 Zn 2 SOD), providing strong evidence for the loss of the water molecule from the catalytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site similar to that in Cu 2 Zn 2 SOD. The reduced type 2 Cu site of AxNiR is shown to be unable to bind inhibitory ligands such as azide, and to react very sluggishly with nitrite leading to only a slow re-oxidation of the the type 1 centre. These observations pro- vide strong evidence that turnover of AxNiR proceeds by an ordered mechanism in which nitrite binds to the oxidised type 2 Cu centres before electron transfer from the reduced type 1 centre occurs. We pro- pose that the two links between the Cu sites of AxNiR, namely His129- Cys130 and His89-Asp92-His94 are utilised for electron transfer and for communicating the status of the type 2 Cu site, respectively. Nitrite bind- ing at type 2 Cu is sensed by the proton abstracting group Asp92 and the type 2 Cu ligand His94, and relayed to the type 1 Cu site via His89 thus triggering an internal electron transfer. The similarity of the type 2 Cu NiR catalytic site to the reduced Cu site of SOD is examined in some detail together with the biochemical evidence for the SOD activity of AxNiR. # 1999 Academic Press Keywords: catalysis; nitrite reductase; superoxide dismutase; EXAFS; electron transfer *Corresponding author Introduction Dissimilatory copper nitrite reductases play an essential role in the cycling of nitrogen in the biosphere, catalysing the reaction NO 2   1e   2H  ! NO  H 2 O (Zumft, 1997). Nitrite reductases are broadly classi®ed into two families, one with haem prosthetic groups and the other with copper centres. Both of these families have attracted much attention and in the last ®ve years several crystallographic structures have been reported. The copper-based nitrite reductases are further sub-divided into two sub-classes primarily due to their colour, namely the green and blue NiRs, thought to be physiological redox partners of pseudo-azurins and azurins, respectively. Crystal- lographic structures of both of the sub-classes have been reported recently (Dodd et al., 1997, 1998; Godden et al., 1991; Kokimota et al., 1994; Adman E-mail address of the corresponding author: s.hasnain@dl.ac.uk Abbreviations used: NiR, nitrite reductase; SOD, superoxide dismutase; ENDOR, electron nuclear double resonance, EXAFS, extended X-ray absorption ®ne structure, EPR, electron paramagnetic resonance. Article No. jmbi.1999.2648 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 287, 1001±1009 0022-2836/99/151001±09 $30.00/0 # 1999 Academic Press