The bioinorganic chemistry of the simple nitrogen oxides (NOx) provides important insights into their environmental and biological functions. Fungi and bacteria utilize heme-containing NO reductase (NOR) enzymes to convert the signaling agent nitric oxide (NO) to the greenhouse gas nitrous oxide (N₂O). Although the overall process of NO–to–N₂O conversion is well-known, experimental systems that model the stepwise chemistry, in particular the critical N–N bond formation steps, of the fungal NOR and bacterial (bac) NOR enzymes remain largely underdeveloped. The Award from the National Science Foundation allowed the collaborative team from the University of Oklahoma (OU: a PhD research institution) and Southern Illinois University Edwardsville (SIUE; a primarily undergraduate institution) to develop experimental systems that provided unique insight into the heme-mediated bioinorganic chemistry of NO of relevance to human health and the environment.

INTELLECTUAL MERIT:  We demonstrated that the function of the di-iron heme/non-heme site in bacNOR could be effectively modeled through the Lewis acid-activation of a heme–NO unit that was not limited to iron (Fe). We showed, for the first time, that the earth-abundant element cobalt was also a viable metal for the heme-mediated conversion of NO to N₂O, although to a lesser extent than Fe. We utilized results of computational calculations to rationalize why Fe is the natural choice for these N–N coupling reactions catalyzed by bacteria. In addition, we demonstrated a then-unprecedented monodentate binding of diazeniumdiolates (NONOates) to a heme model. As NONOates are commonly utilized as NO donors, our demonstration of monodentate binding opens up new avenues for studying the heme-mediated biocoordination chemistry of NONOates and their reactivity as a function of binding mode.

Nitrosoarenes (ArN=O; Ar = aryl) and nitrosoalkanes (RN=O; R = alkyl) are oxidative and reductive metabolites that bind competitively to heme proteins and essentially shut down their function by binding to their Fe centers to prevent oxygen binding. We provided, for the first time, using a combination of experiment and computational chemistry, a molecular orbital rationale for the experimentally observed N-binding, rather than the alternate O-binding, of ArNO molecules to the ferrous centers of heme proteins. We elucidated the specific molecular orbital interactions between the Fe and ArNO atoms that lead to stable binding in the ferrous state but not in the oxidized/ferric state. We extended our synthetic work to the prototypical heme protein myoglobin (Mb) and its interactions with ArNO and RNO ligands. We successfully crystallized and demonstrated, through the use of protein crystallography, this N-binding mode of various nitrosoalkanes to wild-type and distal pocket His64Ala mutant Mbs. We showed that although distal pocket residues may affect the orientation and conformation of the bound RN=O ligands, the N-binding mode for the ferrous heme sites was retained throughout. Oxidation of these Mb-RNO complexes to their ferric forms resulted in displacement of the RN=O ligands with H₂O molecules.

Amphetamine and its oxidative metabolite N-hydroxyamphetamine (AmphNHOH) are biologically active molecules, with AmphNHOH subsequently binding to and inhibiting heme proteins. We showed that the blood protein hemoglobin (Hb) interacts with the AmphNHOH metabolite to generate the nitroso form AmphNO that, despite its large size, binds directly to the Fe center of Hb. Our structural biology approach revealed protein chain movements that allowed the AmphNO ligand to be accommodated with the heme protein distal pocket active site.  We also showed, through the use of synthetic chemistry and computational calculations, that the AmpNHOH molecule is specifically activated by a heme macrocycle (i.e., in addition to binding to the Fe center) through a hitherto unrecognized pi-to-sigma* interaction from a porphyrin N=C donor group to the acceptor sigma* orbital of the –OH moiety of AmphNHOH. This pi-to-sigma* interaction likely activates the hydroxyl O–H bond towards further reactivity to yield the nitroso product.

BROADER IMPACTS: This collaborative grant involving an R1 institution (OU, the PI) and a PUI (SIUE, co-PI) allowed us to significantly improve the knowledge surrounding the heme-mediated chemistry of NO and related molecules. We further modernized our educational tools for the training of students. As part of this effort, we significantly improved a new freshman General Chemistry course at the PI's institution to include a lecture and lab on computational chemistry. The course materials continued to be shared with other faculty in various institutions across the country. The co-PI's development of electrochemistry educational materials continued to play a key role in our broader impacts and outreach to the larger community.

Research results were discussed in the context of online virtual lectures on popular science topics through the Science Circle (SC) Foundation in Second Life (SL); archived recordings of these lectures developed by the co-PI are available on YouTube.

Eleven graduate students and five undergraduates participated in this collaborative grant, as did one postdoctoral fellow. Peer-reviewed manuscripts were published, and the work was disseminated at several regional and national conferences.

Last Modified: 10/04/2022
Modified by: Ann H West