The multicopper oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity.
The multicopper oxidase from the hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) was overproduced in Escherichia coli and purified to homogeneity. The enzyme consists of a single 49.6 kDa subunit, and the combined results of UV-visible, CD, EPR and resonance Raman spectroscopies showed the characteristic features of the multicopper oxidases. Analysis of the McoP sequence allowed its structure to be derived by comparative modeling methods. This model provided a criterion for designing meaningful site-directed mutants of the enzyme. McoP is a hyperthermoactive and thermostable enzyme with an optimum reaction temperature of 85 degrees C, a half-life of inactivation of approximately 6 h at 80 degrees C, and temperature values at the midpoint from 97 to 112 degrees C. McoP is an efficient metallo-oxidase that catalyzes the oxidation of cuprous and ferrous ions with turnover rate constants of 356 and 128 min(-1), respectively, at 40 degrees C. It is noteworthy that McoP follows a ping-pong mechanism, with three-fold higher catalytic efficiency when using nitrous oxide as electron acceptor than when using dioxygen, the typical oxidizing substrate of multicopper oxidases. This finding led us to propose that McoP represents a novel archaeal nitrous oxide reductase that is most probably involved in the final step of the denitrification pathway of P. aerophilum.
Fernandes
André T
AT
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
Damas
João M
JM
Todorovic
Smilja
S
Huber
Robert
R
Baratto
M Camilla
MC
Pogni
Rebecca
R
Soares
Cláudio M
CM
Martins
Lígia O
LO
D000595:Amino Acid Sequence; D001105:Archaea; D003001:Cloning, Molecular / Q000379:methods; D003300:Copper / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D041543:Pyrobaculum / Q000201:enzymology*; D013057:Spectrum Analysis; D013696:Temperature
D003300:Copper; D007501:Iron; D010088:Oxidoreductases; C024748:copper oxidase; C022763:nitrous oxide reductase