A model of the copper centres of nitrous oxide reductase (Pseudomonas stutzeri)

Abstract

Nitrous oxide reductase (N2OR), Pseudomonas stutzeri, catalyses the 2 electron reduction of nitrous oxide to di‐nitrogen. The enzyme has 2 identical subunits (M 1 ∼ 70 000) of known amino acid sequence and contains ∼ 4 Cu ions per subunit. By measurement of the optical absorption, electron paramagnetic resonance (EPR) and low‐temperature magnetic circular dichroism (MCD) spectra of the oxidised state, a semi‐reduced form and the fully reduced state of the enzyme it is shown that the enzyme contains 2 distinct copper centres of which one is assigned to an electron‐transfer function, centre A, and the other to a catalytic site, centre Z. The latter is a binuclear copper centre with at least 1 cysteine ligand and cycles between oxidation levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or inhibitors. The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra provide evidence for a second form of centre Z, which may be enzymatically active, in the oxidised state of the enzyme. Centre A is structurally similar to that of CuA in bovine and bacterial cytochrome c oxidase and also contains copper ligated by cysteine. This centre may also be a binuclear copper complex.

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

“…Although the presence of the CuZ center was proposed about 20 years ago [43], our knowledge of its chemistry is still scarce and owing to its characteristics it represents a fascinating chapter in the field of bioinorganic chemistry.…”

“…Since it was considered that the CuA center was the electron transfer site (like in COX), this other copper center was proposed to be the catalytic center, and was named by Farrar et al [43] as the CuZ center. At that time, the CuZ center was proposed to be a binuclear copper center with properties different from those of either a type 3 copper center or a CuA center.…”

“…The EPR spectrum of the ''as-isolated,'' purple form (Table 1, form A) exhibits a well-defined seven-line hyperfine splitting, whereas that of the dithionite-reduced form (Table 1, form C) has a broad and poorly resolved four-line hyperfine splitting signal [43]. However, the ''semireduced'' form (Table 1, form B), obtained by reducing the ''purple'' form with sodium ascorbate, shows an extremely weak EPR signal and must be considered EPR-silent, but still has an absorption band at 540 nm.…”

“…Farrar et al [43] suggested the presence of two distinct forms of the CuZ center, named CuZ and CuZ*. In particular, the catalytic center can exist in a redox-active form, CuZ, and in a redox-inactive form, CuZ*, in which the CuZ center is [2Cu 2?…”

“…It was also noticed that the amount of CuZ* species was dependent on the N 2 OR isolation procedure. It was shown that the proportion of CuZ* is minimal in Pseudomonas stutzeri N 2 OR isolated under the exclusion of oxygen [43]. In fact, the Paracoccus pantotrophus N 2 OR purified under the exclusion of oxygen has a CuZ* to total CuZ ratio of 0.29, whereas in the preparation in the presence of oxygen, this ratio is 0.66 [68].…”