Structural and kinetic evidence for an ordered mechanism of copper nitritereductase

The crystallographic structures of several copper-containing nitrite reduct ases are now available. Despite this wealth of structural data, no definiti ve information is available as to whether the reaction proceeds by an order ed mechanism where nitrite binds to the oxidised type 2 site, followed by a n 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 first structural information on both types of Cu centres for the reduced form of NiR from Alcaligenes xylosoxidans (AxNiR) using X-ray absor ption spectroscopy. The reduced type 2 Cu site EXAFS shows striking similar ity to the EXAFS data for reduced bovine superoxide dismutase (Cu,Zn, SOD), providing strong evidence for the loss of the water molecule from the cata lytic Cu site in NiR on reduction resulting in a tri-coordinate Cu site sim ilar to that in Cu,Zn, 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 slug gishly with nitrite leading to only a slow re-oxidation of the the type 1 c entre. These observations provide strong evidence that turnover of AxNiR pr oceeds 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 propose that the two links between the Cu sites of AxNiR, namely His12 9-Cysl30 and His89-Asp92-His94 are utilised for electron transfer and for c ommunicating the status of the type 2 Cu site, respectively. Nitrite bindin g 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 trigge ring an internal electron transfer. The similarity of the type 2 Cu NiR cat alytic site to the reduced Cu site of SOD is examined in some detail togeth er with the biochemical evidence for the SOD activity of AxNiR. (C) 1999 Ac ademic Press.