Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617.
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.
Prudêncio
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Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2825-114 Caparica, Portugal.
Pereira
A S
AS
Tavares
P
P
Besson
S
S
Cabrito
I
I
Brown
K
K
Samyn
B
B
Devreese
B
B
Van Beeumen
J
J
Rusnak
F
F
Fauque
G
G
Moura
J J
JJ
Tegoni
M
M
Cambillau
C
C
Moura
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D003300:Copper; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D011549:Pseudomonas / Q000737:chemistry* / Q000201:enzymology*; D016415:Sequence Alignment
D003300:Copper; D010088:Oxidoreductases; C022763:nitrous oxide reductase