Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N 2 O). It has been proposed that N 2 O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH 2 OH) quantitatively to N 2 O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH 2 OH to nitrite (NO − 2 ) under aerobic conditions. Although we observe NO − 2 formation under aerobic conditions, its concentration is not stoichiometric with the NH 2 OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH 2 OH to N 2 O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe III -NH 2 OH adduct of cyt P460 is oxidized to an {FeNO} 6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N-N bond of N 2 O during a bimolecular, rate-determining step. We propose that NO − 2 results when nitric oxide (NO) dissociates from the {FeNO} 6 intermediate and reacts with dioxygen. Thus, NO − 2 is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH 2 OH contributes to NO and N 2 O emissions from nitrifying microorganisms. possesses a global warming potential nearly 300-fold greater than carbon dioxide (1). Atmospheric N 2 O concentrations have increased ∼120% since the preindustrial era, largely due to the widespread use of fertilizers required to produce sustenance for humans and livestock. N 2 O is a byproduct of the microbial metabolism of fertilizer components, including ammonia (NH 3 ) and nitrate (NO − 3 ); consequently, agricultural soils account for an estimated 60-75% of global N 2 O emissions. The metabolic pathway by which microorganisms oxidize NH 3 , nitrification, occurs in two phases, both of which are mediated by autotrophic microorganisms. In the first, NH 3 -oxidizing bacteria (AOB) or archaea (AOA) oxidize NH 3 to nitrite (NO − 2 ). In the second, NO − 2 is subsequently oxidized to NO − 3 by NO − 2 -oxidizing bacteria. NH 3 -oxidizing microbes contribute substantially to global N 2 O emissions, whereas NO − 2 -oxidizing bacteria produce negligible N 2 O (2, 3). AOB are proposed to emit N 2 O either as a byproduct of the nitrification pathway or as a product of the nitrifier denitrification pathway (i.e., the reduction of NO − 2 ) (4-6). Nitrification of NH 3 to NO − 2 occurs in two steps (7,8). The first step is catalyzed by NH 3 monooxygenase, which uses copper (Cu) and dioxygen (O 2 ) to hydroxylate NH 3 to hydroxylamine (NH 2 OH) (9). In AOB, the second step is thought to be the four-electron oxidation of NH 2 OH to NO − 2 by NH 2 OH oxidoreductase (HAO). HAO is a multiheme enzyme with eight c-type hemes per subunit: seven are electron transfer cofactors, and the eighth is the so-called P460 active site that contains a unique tyrosine cross-link to the ...

A vital role has been identified for the heme-lysine cross-link unique to cytochromes P460: preventing enzyme deactivation during catalysis by the obligate nitrification metabolite nitric oxide.

One amino acid makes the difference between a metalloenzyme and a metalloprotein in two otherwise effectively identical cytochrome P460s.

The synthesis and characterization of a Co(II) dithiolato complex Co(M e3 TACN)(S 2 SiMe 2 ) (1) is reported. Reaction of 1 with O 2 generates a rare thiolate-ligated cobalt-superoxo species Co(O 2 ) (M e3 TACN)(S 2 SiMe 2 ) (2) that was characterized spectroscopically and structurally by resonance Raman, EPR, and X-ray absorption spectroscopies as well as density functional theory (DFT). Metal-superoxo species are proposed to S-oxygenate metal-bound thiolate donors in nonheme thiol dioxygenases, but 2 does not lead to S-oxygenation of the intramolecular thiolate donors, and does not react with exogenous sulfur donors. However, complex 2 is capable of oxidizing the O-H bonds of 2,2,6,6-tetramethylpiperidin-1-ol (TEMPOH) derivatives via H-atom abstraction. Complementary proton-coupled electron-transfer (PCET) reactivity is seen for 2 with separated proton/reductant pairs. The reactivity studies indicate that 2 can abstract H-atoms from weak X-H bonds with BDFE ≤ 70 kcal mol-1. DFT calculations predict that the putative Co(OOH) product has an O-H bond dissociation free energy (BDFE) = 67 kcal mol −1 , which matches the observed pattern of reactivity seen for 2. These data provide new information regarding the selectivity of Soxygenation versus H-atom abstraction in thiolate-ligated nonheme metalloenzymes that react with O 2 .

The activation of dioxygen by FeII(Me3TACN)­(S2SiMe2) (1) is reported. Reaction of 1 with O2 at −135 °C in 2-MeTHF generates a thiolate-ligated (peroxo)­diiron complex FeIII 2(O2)­(Me3TACN)2(S2SiMe2)2 (2) that was characterized by UV–vis (λmax = 300, 390, 530, 723 nm), Mössbauer (δ = 0.53, |ΔE Q| = 0.76 mm s–1), resonance Raman (RR) (ν­(O–O) = 849 cm–1), and X-ray absorption (XAS) spectroscopies. Complex 2 is distinct from the outer-sphere oxidation product 1 ox (UV–vis (λmax = 435, 520, 600 nm), Mössbauer (δ = 0.45, |ΔE Q| = 3.6 mm s–1), and EPR (S = 5/2, g = [6.38, 5.53, 1.99])), obtained by one-electron oxidation of 1. Cleavage of the peroxo O–O bond can be initiated either photochemically or thermally to produce a new species assigned as an FeIV(O) complex, FeIV(O)­(Me3TACN)­(S2SiMe2) (3), which was identified by UV–vis (λmax = 385, 460, 890 nm), Mössbauer (δ = 0.21, |ΔE Q| = 1.57 mm s–1), RR (ν­(FeIVO) = 735 cm–1), and X-ray absorption spectroscopies, as well as reactivity patterns. Reaction of 3 at low temperature with H atom donors gives a new species, FeIII(OH)­(Me3TACN)­(S2SiMe2) (4). Complex 4 was independently synthesized from 1 by the stoichiometric addition of a one-electron oxidant and a hydroxide source. This work provides a rare example of dioxygen activation at a mononuclear nonheme iron­(II) complex that produces both FeIII–O–O–FeIII and FeIV(O) species in the same reaction with O2. It also demonstrates the feasibility of forming Fe/O2 intermediates with strongly donating sulfur ligands while avoiding immediate sulfur oxidation.

Coordination of redox-active ligands to metals is a compelling strategy for making reduced complexes more accessible. In this work, we explore the use of redox-active formazanate ligands in low-coordinate iron chemistry. Reduction of an iron(II) precursor occurs at milder potentials than analogous non-redox-active β-diketiminate complexes, and the reduced three-coordinate formazanate-iron compound is characterized in detail. Structural, spectroscopic, and computational analysis show that the formazanate ligand undergoes reversible ligand-centered reduction to form a formazanate radical dianion in the reduced species. The less negative reduction potential of the reduced low-coordinate iron formazanate complex leads to distinctive reactivity with formation of a new N-I bond that is not seen with the β-diketiminate analogue. Thus, the storage of an electron on the supporting ligand changes the redox potential and enhances certain reactivity.

Cytochrome (cyt) P460 is a c-type monoheme enzyme found in ammonia-oxidizing bacteria (AOB) and methanotrophs; additionally, genes encoding it have been found in some pathogenic bacteria. Cyt P460 is defined by a unique post-translational modification to the heme macrocycle, where a lysine (Lys) residue covalently attaches to the 13′ meso carbon of the porphyrin, modifying this heme macrocycle into the enzyme’s eponymous P460 cofactor, similar to the cofactor found in the enzyme hydroxylamine oxidoreductase. This cross-link imbues the protein with unique spectroscopic properties, the most obvious of which is the enzyme’s green color in solution. Cyt P460 from the AOB Nitrosomonas europaea is a homodimeric redox enzyme that produces nitrous oxide (N2O) from 2 equiv of hydroxylamine. Mutation of the Lys cross-link results in spectroscopic features that are more similar to those of standard cyt c′ proteins and renders the enzyme catalytically incompetent for NH2OH oxidation. Recently, the necessity of a second-sphere glutamate (Glu) residue for redox catalysis was established; it plausibly serves as proton relay during the first oxidative half of the catalytic cycle. Herein, we report the first crystal structure of a cross-link deficient cyt P460. This structure shows that the positioning of the catalytically essential Glu changes by approximately 0.8 Å when compared to a cross-linked, catalytically competent cyt P460. It appears that the heme–Lys cross-link affects the relative position of the P460 cofactor with respect to the second-sphere Glu residue, therefore dictating the catalytic competency of the enzyme.

Reaction of the mononuclear nonheme complex [FeII(CH3CN)(N3PyS)]BF4 (1) with an HNO donor, Piloty’s acid (PhSO2NHOH, P.A.), at low temperature affords a high-spin (S = 2) FeII-P.A. intermediate (2), characterized by 57Fe Mössbauer and Fe K- edge X-ray absorption (XAS) spectroscopies, with interpretation of both supported by DFT calculations. The combined methods indicate that P.A. anion binds as the N-deprotonated tautomer (PhSO2NOH−) to [FeII(N3PyS)]+, leading to 2. Complex 2 is the first spectroscopically characterized example, to our knowledge, of P.A. anion bound to a redox-active metal center. Warming of 2 above −60 °C yields the stable {FeNO}7 complex [Fe(NO)(N3PyS)]BF4 (4), as evidenced by 1H NMR, ATR-IR, and Mössbauer spectroscopies. Isotope labeling experiments with 15N-labeled P.A. confirm that the nitrosyl ligand in 4 derives from P.A. In contrast, addition of a second equivalent of a strong base leads to S-N cleavage and production of an {FeNO}8 species, the deprotonated analog of an Fe-HNO complex. This work has implications for the targeted delivery of HNO/ NO−/NO‧ to nonheme Fe centers in biological and synthetic applications, and suggests a new role for nonheme FeII complexes in the assisted degradation of HNO donor molecules.