Isotope Effects Associated with N2O Production by Fungal and Bacterial Nitric Oxide Reductases: Implications for Enzyme Mechanisms

Abstract:

Nitrous oxide (N₂O) is both a powerful greenhouse gas and a key participant in ozone destruction. Microbial activity accounts for over 70% of the N₂O produced annually, and the atmospheric concentration of N₂O continues to rise. Because the fungal and bacterial denitrification pathways are major contributors to microbial N₂O production, understanding the mechanism by which NO is reduced to N₂O will contribute to both N₂O source tracing and quantification. Our strategy utilizes stable isotopes to probe the enzymatic mechanism of microbial N₂O production. Although the use of stable isotopes to study enzyme mechanisms is not new, our approach is distinct in that we employ both measurements of isotopic preferences of purified enzyme and DFT calculations, thereby providing a synergistic combination of experimental and computational approaches.

We analyzed δ¹⁸O, δ¹⁵N^α (central N atom in N₂O), and δ¹⁵N^β (terminal N atom) of N₂O produced by purified fungal cytochrome P450 nitric oxide reductase (P450nor) from Histoplasma capsulatum as well as bacterial cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans. P450nor exhibits an inverse kinetic isotope effect for N^β (KIE = 0.9651) but a normal isotope effect for both N^α (KIE = 1.0127) and the oxygen atom (KIE = 1.0264). These results suggest a mechanism where NO binds to the ferric heme in the P450nor active site and becomes N^β. Analysis of the NO-binding step indicated a greater difference in zero point energy in the transition state than the ground state, resulting in the inverse KIE observed for N^β. Following protonation and rearrangement, it is speculated that this complex forms a Fe^IV-NHOH^- species as a key intermediate. Our data are consistent with the second NO (which becomes N^α and O in the N₂O product) attacking the Fe^IV-NHOH^- species to generate a Fe^III-N₂O₂H₂ complex that enzymatically (as opposed to abiotically) breaks down to release N₂O. Conversely, our preliminary data indicate that cNOR exhibits an inverse isotope effect for both N^α and N^β, but a normal isotope effect for the oxygen. These results are consistent with a very different mechanism for the bimetallic active site in bacterial cNOR in which one NO binds to a non-heme Fe_B site while the other NO binds to the heme b₃ site.