In microbial denitrification process, nitrate (NO3−
), which is the main nitrogen-containing pollutant in ground water, is stepwise reduced to harmless dinitrogen (N2
) thorough nitrite (NO2−
), nitric oxide (NO) and nitrous oxide (N2
O). Although ideally no intermediate metabolite is lost to the environment, certain soil bacteria release N2
O into the atmosphere. N2
O is known to contribute to the green house gas effect and to ozone depletion. The largest emission source of N2
O is bacterial breakdown of nitrogen compounds and the key enzyme in the N2
O production is a metalloenzyme of nitric oxide reductase (NOR). NOR is a membrane-integrated protein and catalyzes the reduction of NO to N2
O at active sites of iron co-factors of the non-heme FeB
and/or the heme b3
.The NO reduction involves the N−O bond cleavage and the N−N formation. The reaction mechanism is interesting from a chemical point of view because the NO reduction is known to govern the selectivity of the final product, N2
, in electrochemical nitrate reduction.Three possible reaction mechanisms are proposed: trans-
mechanism and cis-
mechanism.However, the molecular mechanism of the NO reduction catalyzed by NOR still remains to be solved. Herein, we report protein film voltammetry (PFV) and surface enhanced infrared absorption (SEIRA) spectroscopy of NOR at gold electrodes to gain mechanistic insights into the NO reduction. PFV allows us to apply electrochemical potentials to redox-active enzymes including NOR on the electrode surface and to initiate electrocatalytic reactions by the enzyme. SEIRA spectroscopy is a powerful technique to understand reaction mechanisms of enzymes at the electrode surface.In this work, a cytochrome c-
dependent NOR was isolated from Pseudomonas Aeruginosa
and then immobilized directly on the surface of a gold-coated Si hemicylinder prism or via self-assembled monolayers for cyclic voltammetry and SEIRA spectroscopy. Cyclic voltammograms of NOR-modified electrodes showed reduction current under NO but not under Ar, suggesting that NOR on the electrode surface catalyzed the NO reduction to N2
O. Potential-dependent SEIRA spectra of NOR-modified electrodes were recorded under NO and characteristic two bands were observed in the range from 1600 cm−1
to 1800 cm−1
. NO molecules bonded to the non-heme FeB
and to the heme b3
are known to show absorption bands in this IR range.Thus, it is most likely that NOR on the electrode may catalyze the NO reduction in the trans mechanism.
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