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 MQH(2): 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 (g(z) = 3.015; g(y) = 2.226; g(x) = 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 (K-i( NO) = 7 muM). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degreesC for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes O-p1 and O-p2, 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 bo(3) and aa(3) oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.