No abstract

Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr[H+]+[H+]2)/(K(0)+[H+]), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.

No abstract

Dissimilatory nitrite reductase (NIR) is a key enzyme in denitrification, catalyzing the first step that leads to gaseous products (NO, N 2O, and N2). We have determined the crystal structure of a Cu-containing NIR from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans, at 2.2-Å resolution. The overall structure of this H. denitrificans NIR reveals a trigonal prism-shaped molecule in which a monomer consisting of 447 residues and three Cu atoms is organized into a unique hexamer (i.e., a tightly associated dimer of trimers). Each monomer is composed of an N-terminal region containing a Greek key ␤-barrel folding domain, cupredoxin domain I, and a C-terminal region containing cupredoxin domains II and III. Both cupredoxin domains I and II bind one type 1 Cu and are combined with a long loop comprising 31 amino acid residues. The type 2 Cu is ligated at the interface between domain II of one monomer and domain III of an adjacent monomer. Between the two trimeric C-terminal regions are three interfaces formed by an interaction between the domains I, and the type 1 Cu in the domain is required for dimerization of the trimer. The type 1 Cu in domain II functions as an electron acceptor from an electron donor protein and then transfers an electron to the type 2 Cu, binding the substrate to reduce nitrite to NO. The discussion of the intermolecular electron transfer process from cytochrome c 550 to the H. denitrificans NIR is based on x-ray crystallographic and kinetic results.denitrification ͉ electron transfer ͉ redox partner ͉ intermolecular interaction ͉ cupredoxin

The crystal structure of the blue nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) has been determined at 2.05 A resolution. AxgNIR contains both type 1 and 2 Cu sites, the geometry of the former being distorted tetrahedral. The superpositioning of the type 1 Cu sites in the blue enzyme and a green nitrite reductase revealed that the orientation of the Met150 side chain differed. The deviation of the Sdelta(Met150) atom from the axial position of the NNS plane formed by two Ndelta(His95 and His145) and one Sgamma(Cys136) atom caused the difference in the colors of the enzymes, i.e. blue and green.