Abstract
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.
Highlights
Denitrification serves as an alternative to aerobic respiration to generate a proton motive force and is found in many prokaryotic microbes that thrive under anaerobic conditions
In P. aerophilum, both the nitrite reductase and nitrous oxide reductase are bound to the membrane, in contrast to Gram-negative bacteria [11, 15]
Two conserved histidines are ligands to a low spin heme center, which serves as the site where electrons enter; one histidine residue acts as a ligand to a high spin heme center, and the remaining three histidine residues coordinate to FeB in the NO reductases or to CuB in the oxidase branch
Summary
Denitrification serves as an alternative to aerobic respiration to generate a proton motive force and is found in many prokaryotic microbes that thrive under anaerobic conditions. Several archaea are capable of denitrification (e.g. the halophiles Haloferax denitrificans and Haloarcula marismortui and the hyperthermophiles Ferroglobus placidus and Pyrobaculum aerophilum) [11,12,13,14]. These organisms were shown to reduce nitrate as in bacteria via nitrite, NO, and N2O to N2. In P. aerophilum, both the nitrite reductase and nitrous oxide reductase are bound to the membrane, in contrast to Gram-negative bacteria [11, 15].
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