SPECTROSCOPIC CHARACTERIZATION AND INTRAMOLECULAR ELECTRON-TRANSFER PROCESSES OF NATIVE AND TYPE-2 CU-DEPLETED NITRITE REDUCTASES

Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying bacteria (Ac hromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015 , and Alcaligenes xylosoxidans GIFU 1051) have been characterized by e lectronic absorption, circular dichroism, and electron paramagnetic re sonance spectra. The characteristic visible absorption spectra of thes e NIRs are due to the type 1 Cu centers, while the type 2 Cu centers h ardly contribute in the same region. The intramolecular electron trans fer (ET) process from the type 1 Cu to the type 2 Cu in native NIRs ha s been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in T2D NIRs exhibits the same reoxid ation. The ET process obeys first-order kinetics, and observed rate co nstants are 1400-1900 s(-1) (t(1/2) = ca. 0.5 ms) at pH 7.0. In the pr esence of nitrite, the ET process also obeys first-order kinetics, wit h rate constants decreased by factors of 1/12-1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V, close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite also would s hift the redox potential of the type 2 Cu by coordination, and hence t he intramolecular ET rate constant is decreased. Pulse radiolysis expe riments on T2D NIRs in the presence of nitrite demonstrate that the ty pe 1 Cu(I) site is slowly oxidized with a first-order rate constant of 0.03 s(-1) at pH 7.0, suggesting that nitrite bound to the protein ac cepts an electron from the type 1 Cu. This result is in accord with th e finding that T2D NIRs show enzymatic activities, although they are l ower than those of the native enzymes.