The pH-Dependent Changes of Intramolecular Electron Transfer on Copper-Containing Nitrite Reductase

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.

pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

“…Thus, the type 2 copper is unable to accept electrons from the type 1 copper site at high pH. This may contribute to the rapid decrease in enzyme activity as the pH is increased from 6.0 to 9.0 (23,24). Similar conclusions were drawn from studies of the change in type 2 copper site reduction potential in the Cu-NiRs from A. xylosoxidans and A. cycloclastes (1).…”

“…All details concerning the catalytic role of His-287 are not entirely clear, but this residue is believed to be involved in supplying the proton needed for the enzymatic reaction to occur (6) and has in Rhodobacter sphaeroides been seen to disorder at high pH, most likely due to loss of H-bond interactions (12). Nitrite reductase is highly pH-dependent and has an activity maximum between pH 5 and 6 (23,24). One property influencing the activity of NiR is the active-site affinity of the substrate, which in turn varies with pH (25).…”

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

“…However, k ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitritebound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).…”

“…Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7,16,25,(27)(28)(29)(30). However, k ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitritebound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).…”

“…Asp-92 and His-249 have been proposed as acid-base catalysts (18,21,22,28,38), and the abrupt drop in rates at increasing pH may indicate that OH Ϫ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35,40,41).…”