Nitrite reductases (NiR) perform integral steps in the biochemical cycling of nitrogen, transforming NO₂^- to a variety of reduced products.  These processes are of major importance in the denitrification of soil fertilizers (a major source of atmospheric N₂ and N₂O) and in the industrial removal of NO_x pollutants from wastewater. We are using electroactive myoglobin as a hemeprotein model of this reactivity.  By following specific reactions electrochemically, we hope to gain an understanding of the stepwise processes which occur in the native enzymes.

Two distinct biological reactivities are known: assimilatory nitrite reductases (aNiR) reduce nitrite to ammonia for incorporation into biomass; dissimilatory nitrite reductases (dNiR) take part in multi-enzyme denitrification processes yielding the N-N coupled gases N₂O and N₂. Recently, specific hemeproteins have been identified which catalyze each step of the transformation from NO₂^- to NO to N₂O to N₂.

We use a new electrochemical technique, developed by the Rusling group at the University of Conneticut, to initiate and follow multi-electron nitrite reduction using myoglobin  as a hemeprotein model.  The idea is to form an electroactive film which incorporates myoglobin on an electrode surface.  For this we use a water-insoluble surfactant, dimethyldidodecylammonium bromide, or ddab.

The electrochemical response of Mb in these films resembles that of an Fe-porphyrin in an organic solvent. The active site is protected from aqueous solution by both the protein and the surfactant film. The Fe^III/II couple is nicely reversible, and there is a second couple tentatively assigned to Fe ^II/I --

But ions in the aqueous solution can readily interact with the Fe-active site. Using the same electrode moved between different salt solutions, the peak shifts indicating the aqueous ligands bind to the redox active heme site within the surfactant film.

We form the modified electrodes by absorbing  < nmol Mb into thin films  of
dimethyldidodecylammonium bromide (ddab) on basal plane pyrolytic graphite surface.

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