Purification, Characterization, and Preliminary Crystallographic Study of Copper-Containing Nitrous Oxide Reductase fromPseudomonas nautica617

Abstract

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

The electron transfer complex between nitrous oxide reductase and its electron donors

“…As already suggested in previous studies [23,43], the sequence alignment shows a high homology between A. cycloclastes N 2 OR and P. denitrificans N 2 OR, consisting of 89% identity, whereas P. nautica N 2 OR has a lower sequence identity relative to the other two enzymes, with 59% identity with P. denitrificans N 2 OR and 60% identity with A. cycloclastes N 2 OR (Fig. S1).…”

A new CuZ active form in the catalytic reduction of N2O by nitrous oxide reductase from Pseudomonas nautica

“…The reaction between preactivated N 2 OR and a stoichiometric amount of N 2 O, monitored by UV-vis spectroscopy, showed a rapid (within 1 s) oxidation of the CuA centre, through the increase of the two characteristic absorption bands at 480 and 540 nm [13], and the development of an absorption band at 680 nm, which has not been reported previously. Then, this 680-nm absorption band slowly (on the order of minutes) shifts to the usual position of 640 nm for the resting enzyme (Fig.…”

“…nautica 617 (also known as Marinobacter hydrocarbonoclasticus 617) cellular extract as previously described [13], with some minor modifications. Enzyme concentrations were determined by the Lowry method [22].…”

“…nautica N 2 OR, in the as-isolated state, contains a 1Cu II /3Cu I redox state with a total spin of , where the unpaired electron is delocalized between two or more copper atoms through the bridging sulfide ion [10][11][12]. This state, which has a typical electronic spectral band at 640 nm and a four-line splitting EPR signature [13,14], cannot be easily reduced or oxidized. The catalytically active, fully reduced form (4Cu I ) can be obtained only after a prolonged incubation with reduced methyl viologen (MV) [15,16].…”

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

“…denitrificans [31], Paracoccus denitrificans [32], Wolinella succinogenes [33], Flexibacter canadensis [34], Achromobacter cycloclastes [35], Pseudomonas aeruginosa [36], Paracoccus pantotrophus (formerly Thiosphaera pantotropha) [37], Thiobacillus denitrificans [38], Alcaligenes xylosoxidans [39,40], Pseudomonas nautica (renamed Marinobacter hydrocarbonoclausticus) [41], and more recently Hyphomicrobium denitrificans [42].…”