Electrochemical models for
heme-catalyzed denitrification

     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 NiR 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.  Specific heme proteins have been identified which catalyze each step of the transformation from NO₂^- to NO to N₂O to N₂.

 There are several different heme cofactors found in the enzymes involved in NOx metabolism, three are shown below.

    We use a new electrochemical technique, developed by the Rusling group at the University of Connecticut, to initiate and follow multi-electron nitrite reduction using myoglobin  as a heme protein 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.  We form the modified electrodes by absorbing  sub nmol quantities of Mb into thin films of dimethyldidodecylammonium bromide (ddab) on basal oriented pyrolytic graphite surface.

      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 assigned to Fe ^II/I

   But ions in the aqueous solution 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.

      This electrochemical method has allowed us to examine specific heme-catalyzed steps in the nitrogen cycle shown below.  Our investigations include synthetic and mechanistic aspects- for example, our electrochemical results suggested that a  key intermediate species, the ferrous HNO-adduct of myoglobin was long-lived under certain conditions.  This enabled us to isolate this unusual species, Lin JACS 2000, and to structurally characterize it, Sulc JBIC 2002.   We have also developed electrochemical model systems for the unusual  siroheme and heme cd₁ enzymes.

        Most recently, we have examined the electrochemistry and catalytic abilities of CYP119, a thermophilic cytochrome P450, and myoglobin to compare the effect of histidine vs. thiolate ligation on several distinct mechanistic steps in the conversion of nitrite to ammonia, Immoos JACS 2004,        

     The most notable difference is in product selectivities, CYP119 transforms nitrite to ammonia in almost 100% under conditions that myoglobin converts less than 15%. We suggest the protein rigidity traps the substrate within the heme pocket, allowing multi-electron reductions of individual substrates to occur.  

     This interpretation was borne out in a subsequent communication, Blair JACS 2004, in which we demonstrated that CYP119 catalyzes the multi-electron decomposition of halocarbons solvents such as carbon tetrachloride to methane. As a thermophile, CYP119  is its stabile up to ca. 90⁰C, and reversible electrochemical response  was observed up to that temperature. Importantly, methane production was much increased at higher temperatures.

 

 

Selected recent publications on protein electrochemistry:

“High Temperature Electrocatalysis Using Thermophilic P450 CYP119: Dehalogenation of CCl₄ to CH₄” Blair, E.; Greaves, J.; Farmer, P.J. J. Am. Chem. Soc. 2004, 126, 8632-8633.

 “Electrocatalytic reductions of nitrite, nitric oxide and nitrous oxide by Cytochrome P450 CYP 119”  Immoos, C.E.; Chou, J.; Bayachou, M.; Blair, E.; Farmer, P.J. J. Am. Chem. Soc. 2004, 126, 4934-4942.  

"Direct Assessment of the Reduction Potential of the [4Fe-4S]^1+/0 Couple of the Nitrogenase Fe Protein from Azotobacter vinelandii”  Guo, M.; Sulc, F.; Ribbe, M.W.; Immoos, C.E.; Farmer, P.J.; Burgess, B.K. J. Amer. Chem. Soc. 2002, 124, 2100-12101.

“Unusual voltammetry of manganese-substituted myoglobin in surfactant film: evidence for two redox pathways” Lin, R.; Immoos, C.; Farmer, P.J. J. Biol. Inorg. Chem. 2000, 5, 738-747.

“Catalytic Two-electron Reductions of N₂O and N₃^- by Myoglobin in Surfactant Film” Bayachou, M.; Elkbir, L.; Farmer, P.J. Inorg. Chem. 2000, 39, 289-293.

"Electrochemical Reduction of NO by Myoglobin in Surfactant Film: Characterization and Reactivity of the Nitroxyl (NO-) Adduct" Bayachou, M.; Lin, R.; Cho, W.; Farmer, P. J. J. Am. Chem. Soc. 1998, 120, 9888-9893.

Selected recent publications on N-oxide chemistry:

“Trapping of Nitroxyl by Deoxy Myoglobin” Sulc, F.; Immoos, C.; Pervitsky, D. Farmer, P.J.  J. Amer. Chem. Soc. 2004, 125, 1096-1101.

"¹H NMR Structure of the Heme Pocket of HNO-Myoglobin" Sulc, F.; Fleischer, E.; Farmer, P.J.; Ma, D.; La Mar, G. J. Biol. Inorg. Chem. 2003, 8, 348-352.

"The reduction potential of nitric oxide (NO) and its importance to NO biochemistry" Bartberger, M.D.; Liu, W.; Ford, E.; Miranda, K.M.;  Switzer, C.; Fukuto, J.M.; Farmer, P.J.; Wink, D.A.; Houk K. N. Proc. Nat. Acad. Sci. 2002, 99, 10958-10963.

"O-Atom Transfer from Nitric Oxide Catalyzed by Fe(TPP)" Lin, R.; Farmer, P.J.  J. Am. Chem. Soc. 2001, 123, 1143 -1150.

“The HNO Adduct of Myoglobin: Synthesis and Characterization” Lin, R.; Farmer, P.J. J. Am. Chem. Soc 2000, 122, 2393 –2394.