Purification and characterization of the MQH2:NO oxidoreductase from the hyperthermophilic archaeon Pyrobaculum aerophilum.
The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.
de Vries
Simon
S
Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands. S.deVries@tnw.tudelft.nl
Strampraad
Marc J F
MJ
Lu
Shen
S
Moënne-Loccoz
Pierre
P
Schröder
Imke
I
D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification*; D041543:Pyrobaculum / Q000201:enzymology*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D006418:Heme; D007501:Iron; D010088:Oxidoreductases; C034747:nitric-oxide reductase