Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Kinetic isotope effects and mechanistic implications for cNOR, an NO reductase with a heme/non-heme iron active site (#415)

Elise D Rivett 1 2 , Clarisse M Finders 1 2 , Joshua A Haslun 1 2 , Hasand Gandhi 2 3 , Maximilian Kahle 4 , Pia Ädelroth 4 , Peggy H Ostrom 2 3 , Nathaniel E Ostrom 2 3 , Eric L Hegg 1 2
  1. Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI, United States
  2. DOE Great Lakes Bioenergy Research Center, Michigan State University, East Lansing, MI, United States
  3. Department of Integrative Biology, Michigan State University, East Lansing, MI, United States
  4. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

Nitrous oxide (N2O) is produced by multiple types of metalloenzymes as part of the global nitrogen cycle via processes such as nitrification and denitrification. Unfortunately, N2O is also a potent greenhouse gas, and currently it is the most significant anthropogenic ozone-depleting substance.1, 2 To better understand the microbial processes that involve N2O, we use stable isotope measurements to study the kinetic isotope effects (KIEs) of metalloenzymes that produce or consume N2O. Recently, we developed an expansion of a widely-used isotopic model that allows for accurate determination of position-specific KIEs for the central (α) and outer (β) N atoms in the linear N2O molecule, allowing us to mine more nuanced mechanistic insight from our isotopic data.3 Here we highlight our research on cytochrome c nitric oxide reductase (cNOR), which converts two NO molecules to one N2O molecule and one water molecule. cNOR has a bimetallic active site comprised of a heme iron and non-heme iron, both of which can bind NO, leading to a decades-long debate about where each substrate molecule binds during catalysis. Our data reveal that both N atoms in N2O synthesized by cNOR are subject to normal KIEs of similar magnitude. Thus, 14N is enriched to a similar extent at the α and β positions of N2O during cNOR-catalyzed N2O synthesis. As NO binding to either heme Fe(II) or non-heme Fe(II) in the active site is expected to produce a normal isotope effect, our data suggest that each NO molecule at least transiently binds a reduced iron center prior to N-N bond formation. This insight qualitatively supports either the trans mechanism (where one NO molecule binds each iron center) or a modified version of the cis-heme b3 mechanism (where formation of a heme-NO complex is followed by the transient binding of a second NO to the non-heme iron). Overall, our data provide important constraints for evaluating catalytic mechanisms proposed for cNOR and highlight the need for additional computational modeling to compare predicted and measured KIEs.

  1. Stocker, T. F., D. Qin, G.-K. Plattner, M. Tignor, S.K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P.M. Midgley (2013) IPCC, 2013: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change, Cambridge, United Kingdom and New York, NY, USA.
  2. Ravishankara, A. R., Daniel, J. S., and Portmann, R. W. (2009) Nitrous oxide (N2O): The dominant ozone-depleting substance emitted in the 21st century, Science (New York, N.Y.) 326, 123-125.
  3. Rivett, E. D., Ma, W., Ostrom, N. E., and Hegg, E. L. (2024) Position-specific kinetic isotope effects for nitrous oxide: a new expansion of the Rayleigh model, Biogeosciences 21, 4549-4567.