Background: Nitrite reduction pathways are critical for biological NO production under hypoxia. Results: The mitochondrial enzyme mARC reduces nitrite to NO using cytochrome b 5 as electron donor. Conclusion: mARC forms an electron transfer chain with NADH, cytochrome b 5 , and cytochrome b 5 reductase to reduce nitrite to NO. Significance: mARC proteins may constitute a new pathway for hypoxic NO production in vivo.

Background: Erythrocytes contribute to nitrite-mediated NO signaling, but the mechanism is unclear. Results: Deoxyhemoglobin accounts for virtually all NO made from nitrite by erythrocytes with no contributions from other proposed pathways. Conclusion: Deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions. Significance: Reduction by deoxyhemoglobin accounts for nitrite-mediated NO signaling in blood mediating vessel tone and platelet function.

The focus of this Forum review highlights work from our own laboratories and those of others in the area of biochemical and biologically inspired inorganic chemistry dealing with nitric oxide (nitrogen monoxide, ·NO (g) ) and its biological roles and reactions. The latter focus is on (i) oxidation of ·NO (g) to nitrate by nitric oxide dioxygenases (NOD's), and (ii) reductive coupling of two molecules of ·NO (g) to give N 2 O (g) . In the former case, NOD's are described and the highlighting of possible peroxynitrite-heme intermediates and consequences of this are given by discussion of recent works with myoglobin and a synthetic heme model system for NOD action. Summaries of recent copper complex chemistries with ·NO (g) and O 2(g) leading to peroxynitrite species are given. The coverage of biological reductive coupling of ·NO (g) deals with bacterial nitric oxide reductases (NOR's) with heme/non-heme diiron active sites, and on heme/Cu oxidases such as cytochrome c oxidase which can mediate the same chemistry. Recent designed protein and synthetic model compound (heme/non-heme diiron or heme/copper) as functional mimics are discussed in some detail. We also highlight examples from the chemical literature, not necessarily involving biologically relevant metal ions, which describe the oxidation of ·NO (g) to nitrate (or nitrite) and possible peroxynitrite intermediates, or reductive coupling of ·NO (g) to give nitrous oxide.

The interactions of nitrogen monoxide (•NO; nitric oxide) with transition metal centers continue to be of great interest, in part due to their importance in biochemical processes. Here, we describe •NO (g) reductive coupling chemistry of possible relevance to that process (i.e., nitric oxide reductase (NOR) biochemistry) which occurs at the heme/Cu active site of cytochrome c oxidases (CcOs). In this report, heme/Cu/•NO (g) activity is studied using 1:1 ratios of heme and copper complex components, (F 8 )Fe (F 8 = tetrakis(2,6-difluorophenyl)porphyrinate(2-)) and [(tmpa)Cu I (MeCN)] + (TMPA = tris(2-pyridylmethyl)amine). The starting point for heme chemistry is the mononitrosyl complex (F 8 )Fe(NO) (λ max = 399 (Soret), 541 nm in acetone). Variable temperature 1 H-and 2 H-NMR spectra reveal a broad peak at δ = 6.05 ppm (pyrrole) at RT, which gives rise to asymmetrically split pyrrole peaks at 9.12 and 8.54 ppm at −80°C. A new heme dinitrosyl species, ( Control reaction chemistry shows that both iron and copper centers are required for the NOR type chemistry observed, and that if acid is not present, half the •NO is trapped as a (F 8 )Fe(NO) complex, while the remaining nitrogen monoxide undergoes copper complex promoted disproportionation chemistry. As part of this study, [(F 8 )Fe III ] SbF 6 was synthesized and characterized by X-ray crystallography, along with EPR (77 K: g = 5.84 and 6.12 in CH 2 Cl 2 and THF, respectively) and variable temperature NMR spectroscopies. These structural and physical properties suggest that at RT this complex consists of an admixture of high and intermediate spin states.

A iron-dinitrosyl species ( 6 L)Fe(NO) 2 (2), generated from nitrogen monoxide (•NO) binding to its related iron(II)-mononitrosyl complex ( 6 L)Fe(NO) (1), efficiently effects reductive coupling of two •NO molecules to release nitrous oxide (N 2 O), when Cu + ion and two equiv acid are added; the heme/ Cu product is [( 6 While there is extensive recent activity in the design and study of discrete heme/copper synthetic complexes which resemble HCO active sites and/or effect dioxygen binding and reduction chemistry, 6,10 there are no cases where a synthetic small molecule heme/Cu complex reacts with •NO to effect reductive coupling leading to nitrous oxide. In this report, we describe such a system, employing the binucleating ligand 6 L and its iron and heme-copper derivatives which have been previously used in our investigations involving O 2 -chemistry. 10a The starting point is the reductive nitrosylation of ( 6 L)Fe III (OH) 11 to straightforwardlygive the iron(II)-nitrosyl compound ( 6 L)Fe(NO) (1) (Scheme 1), possessing the expected threeline hyperfine split EPR spectrum 12 (Fig. 1a). 13 As has been reported previously, 14 such complexes may react with additional •NO (g) to form a dinitrosyl species. This occurs here and evidence in support of this formulation, ( 6 L)Fe(NO) 2 are as follows: (a) As monitored by UVvis spectroscopy in acetone (−80 °C), 1 reversibly binds •NO (g) to form ( 6 L)Fe(NO) 2 (2); 2 is stable, but loses •NO (g) upon warming to RT. (b) With formation of 2 at −80 °C, the EPR signal due to ( 6 L)Fe-(NO) (1) disappears, as would be expected for 2, having an even number of electrons. (c) Titration of a −80 °C solution of ( 6 L)Fe(NO) 2 (2) with one equiv (F 8 )Fe II {F 8 = tetrakis(2,6-difluorophenyl)porphyrinate(2-)} leads to a clean conversion to ( 6 L)Fe(NO) (1) plus (F 8 )Fe(NO) (X-Ray structure determined); 2 possesses two equiv of bound •NO. 13The existence of ( 6 L)Fe(NO) 2 (2) . This is based on the observed UV-vis spectrum, appearing as a single species with λ max = 396, 515 nm in THF, matching that of a typical (porphyrinate)Fe III -X high-spin complex (X = a non-coordinating anion like PF 6 − or here B(C 6 F 5 ) 4 − ). Further supporting this formulation, an EPR spectrum (Fig. 1b) reveals that a high-spin heme-Fe III along with a Cu II (tetragonal complex) are both present. 13,16Wang et al.