TY - JOUR
T1 - Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617
AU - Prudêncio, Miguel
AU - Pereira, Alice S.
AU - Tavares, Pedro
AU - Besson, Stéphane
AU - Cabrito, Inês
AU - Brown, Kieron
AU - Samyn, Bart
AU - Devreese, Bart
AU - Van Beeumen, Jozef
AU - Rusnak, Frank
AU - Fauque, Guy
AU - Moura, José J. G.
AU - Tegoni, Mariella
AU - Cambillau, Christian
AU - Moura, Isabel
PY - 2000/4/11
Y1 - 2000/4/11
N2 - 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 ~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 ~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 , Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu(Z) remains reduced (S = 1/2 ). Complete crystallographic data at 2.4 Å 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.
AB - 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 ~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 ~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 , Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu(Z) remains reduced (S = 1/2 ). Complete crystallographic data at 2.4 Å 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.
UR - http://www.scopus.com/inward/record.url?scp=0039619862&partnerID=8YFLogxK
U2 - 10.1021/bi9926328
DO - 10.1021/bi9926328
M3 - Article
C2 - 10747777
AN - SCOPUS:0039619862
SN - 0006-2960
VL - 39
SP - 3899
EP - 3907
JO - Biochemistry
JF - Biochemistry
IS - 14
ER -