Conserved C-Terminal Tail Is Responsible for Membrane Localization and Function of Pseudomonas aeruginosa Hemerythrin.

Many bacteria have hemerythrin (Hr) proteins that bind O2, including Pseudomonas aeruginosa, in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C-terminus relative to a well-characterized Hr from Methylococcus capsulatus, and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C-terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a MhrW143* variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. MhrL112Y, a variant that has a much longer-lived O2-bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both MhrW143* and MhrL112Y had secondary structures, stabilities, and O2-binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C-terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O2-binding.

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes.

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

Spectroscopic Characterization of a Diferric Mycobacterial Hemerythrin-Like Protein with Unprecedented Reactivity toward Nitric Oxide.

Hemerythrin-like proteins (HLPs) are broadly distributed across taxonomic groups and appear to play highly diverse functional roles in prokaryotes. Mycobacterial HLPs contribute to the survival of these pathogenic bacteria in mammalian macrophages, but their modes of action remain unclear. A recent crystallographic characterization of Mycobacterium kansasii HLP (Mka-HLP) revealed the unexpected presence of a tyrosine sidechain (Tyr54) near the coordination sphere of one of the two iron centers. Here, we show that Tyr54 is a true ligand to the Fe2(III) ion which, in conjunction with the presence of a µ-oxo group bridging the two iron(III), brings unique reactivity toward nitric oxide (NO). Monitoring the titration of Mka-HLP with NO by Fourier-transform infrared and electron paramagnetic resonance spectroscopies shows that both diferric and diferrous forms of Mka-HLP accumulate an uncoupled high-spin and low-spin {FeNO}7 pair. We assign the reactivity of the diferric protein to an initial radical reaction between NO and the µ-oxo bridge to form nitrite and a mixed-valent diiron center that can react further with NO. Amperometric measurements of NO consumption by Mka-HLP confirm that this reactivity can proceed at low micromolar concentrations of NO, before additional NO consumption, supporting a NO scavenging role for mycobacterial HLPs.

Recent Insights into the Diversity and Evolution of Invertebrate Hemerythrins and Extracellular Globins.

There are three broad groups of oxygen-transport proteins found in the haemolymph (blood) of invertebrates, namely the hemocyanins, the hemerythrins and the globins. Both hemerythrins and extracellular globins are iron-based proteins that are understudied when compared to the copper-containing hemocyanins. Recent evidence suggests that hemerythrins and (giant) extracellular globins (and their linker chains) are more widely distributed than previously thought and may have biological functions beyond oxygen transport and storage. Herein, we review contemporary literature of these often-neglected proteins with respect to their structural configurations on formation and ancestral states.

Identification and Characterization of a Redox Sensor Phosphodiesterase from Ferrovum sp. PN-J185 Containing Bacterial Hemerythrin and HD-GYP Domains.

The second messenger bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates numerous important physiological functions in bacteria. In this study, we identified and characterized the first dimeric, full-length, non-heme iron-bound phosphodiesterase (PDE) containing bacterial hemerythrin and HD-GYP domains (Bhr-HD-GYP). We found that the amino acid sequence encoded by the FV185_09380 gene from Ferrovum sp. PN-J185 contains an N-terminal bacterial hemerythrin domain and a C-terminal HD-GYP domain, which is characteristic of proteins with PDE activity toward c-di-GMP. Inductively coupled plasma optical emission spectroscopy analyses showed that Bhr-HD-GYP contains 4 equiv of iron atoms per subunit, suggesting both hemerythrin and HD-GYP domains have non-heme di-iron sites. A redox-dependent spectral change expected for oxo-bridged non-heme iron with carboxylate ligands was observed, and this redox interconversion was reversible. However, unlike marine invertebrate hemerythrin, which functions as an oxygen-binding protein, Bhr-HD-GYP did not form an oxygen adduct because of rapid autoxidation. The reduced ferrous iron complex of the protein catalyzed the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas the oxidized ferric iron complex had no significant activity. These results suggest that Bhr-HD-GYP is a redox and oxygen sensor enzyme that regulates c-di-GMP levels in response to changes in cellular redox status or oxygen concentration. Our study may lead to an improved understanding of the physiology of iron-oxidizing bacterium Ferrovum sp. PN-J185.

Crystal structure of a hemerythrin-like protein from Mycobacterium kansasii and homology model of the orthologous Rv2633c protein of M. tuberculosis.

Pathogenic and opportunistic mycobacteria have a distinct class of non-heme di-iron hemerythrin-like proteins (HLPs). The first to be isolated was the Rv2633c protein, which plays a role in infection by Mycobacterium tuberculosis (Mtb), but could not be crystallized. This work presents the first crystal structure of an ortholog of Rv2633c, the mycobacterial HLP from Mycobacterium kansasii (Mka). This structure differs from those of hemerythrins and other known HLPs. It consists of five α-helices, whereas all other HLP domains have four. In contrast with other HLPs, the HLP domain is not fused to an additional protein domain. The residues ligating and surrounding the di-iron site are also unique among HLPs. Notably, a tyrosine occupies the position normally held by one of the histidine ligands in hemerythrin. This structure was used to construct a homology model of Rv2633c. The structure of five α-helices is conserved and the di-iron site ligands are identical in Rv2633c. Two residues near the ends of helices in the Mka HLP structure are replaced with prolines in the Rv2633c model. This may account for structural perturbations that decrease the solubility of Rv2633c relative to Mka HLP. Clusters of residues that differ in charge or polarity between Rv2633c and Mka HLP that point outward from the helical core could reflect a specificity for potential differential interactions with other protein partners in vivo, which are related to function. The Mka HLP exhibited weaker catalase activity than Rv2633c. Evidence was obtained for the interaction of Mka HLP irons with nitric oxide.

Variable-Temperature ESI-IMS-MS Analysis of Myohemerythrin Reveals Ligand Losses, Unfolding, and a Non-Native Disulfide Bond.

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from ∼15 to 92 °C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from ∼15 to 35 °C, some bound oxygen dissociates; at ∼66 °C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (∼85 °C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.

Structure, function and evolution of the hemerythrin-like domain superfamily.

Hemerythrin-like proteins have generally been studied for their ability to reversibly bind oxygen through their binuclear nonheme iron centers. However, in recent years, it has become increasingly evident that some members of the hemerythrin-like superfamily also participate in many other biological processes. For instance, the binuclear nonheme iron site of YtfE, a hemerythrin-like protein involved in the repair of iron centers in Escherichia coli, catalyzes the reduction of nitric oxide to nitrous oxide, and the human F-box/LRR-repeat protein 5, which contains a hemerythrin-like domain, is involved in intracellular iron homeostasis. Furthermore, structural data on hemerythrin-like domains from two proteins of unknown function, PF0695 from Pyrococcus furiosus and NMB1532 from Neisseria meningitidis, show that the cation-binding sites, typical of hemerythrin, can be absent or be occupied by metal ions other than iron. To systematically investigate this functional and structural diversity of the hemerythrin-like superfamily, we have collected hemerythrin-like sequences from a database comprising fully sequenced proteomes and generated a cluster map based on their all-against-all pairwise sequence similarity. Our results show that the hemerythrin-like superfamily comprises a large number of protein families which can be classified into three broad groups on the basis of their cation-coordinating residues: (a) signal-transduction and oxygen-carrier hemerythrins (H-HxxxE-HxxxH-HxxxxD); (b) hemerythrin-like (H-HxxxE-H-HxxxE); and, (c) metazoan F-box proteins (H-HExxE-H-HxxxE). Interestingly, all but two hemerythrin-like families exhibit internal sequence and structural symmetry, suggesting that a duplication event may have led to the origin of the hemerythrin domain.

Broad Phylogenetic Occurrence of the Oxygen-Binding Hemerythrins in Bilaterians.

Animal tissues need to be properly oxygenated for carrying out catabolic respiration and, as such, natural selection has presumably favored special molecules that can reversibly bind and transport oxygen. Hemoglobins, hemocyanins, and hemerythrins (Hrs) fulfill this role, with Hrs being the least studied. Knowledge of oxygen-binding proteins is crucial for understanding animal physiology. Hr genes are present in the three domains of life, Archaea, Bacteria, and Eukaryota; however, within Animalia, Hrs has been reported only in marine species in six phyla (Annelida, Brachiopoda, Priapulida, Bryozoa, Cnidaria, and Arthropoda). Given this observed Hr distribution, whether all metazoan Hrs share a common origin is circumspect. We investigated Hr diversity and evolution in metazoans, by employing in silico approaches to survey for Hrs from of 120 metazoan transcriptomes and genomes. We found 58 candidate Hr genes actively transcribed in 36 species distributed in 11 animal phyla, with new records in Echinodermata, Hemichordata, Mollusca, Nemertea, Phoronida, and Platyhelminthes. Moreover, we found that "Hrs" reported from Cnidaria and Arthropoda were not consistent with that of other metazoan Hrs. Contrary to previous suggestions that Hr genes were absent in deuterostomes, we find Hr genes present in deuterostomes and were likely present in early bilaterians, but not in nonbilaterian animal lineages. As expected, the Hr gene tree did not mirror metazoan phylogeny, suggesting that Hrs evolutionary history was complex and besides the oxygen carrying capacity, the drivers of Hr evolution may also consist of secondary functional specializations of the proteins, like immunological functions.

Discovery and evolution of novel hemerythrin genes in annelid worms.

BACKGROUND: Despite extensive study on hemoglobins and hemocyanins, little is known about hemerythrin (Hr) evolutionary history. Four subgroups of Hrs have been documented, including: circulating Hr (cHr), myohemerythrin (myoHr), ovohemerythrin (ovoHr), and neurohemerythrin (nHr). Annelids have the greatest diversity of oxygen carrying proteins among animals and are the only phylum in which all Hr subgroups have been documented. To examine Hr diversity in annelids and to further understand evolution of Hrs, we employed approaches to survey annelid transcriptomes in silico. RESULTS: Sequences of 214 putative Hr genes were identified from 44 annelid species in 40 different families and Bayesian inference revealed two major clades with strong statistical support. Notably, the topology of the Hr gene tree did not mirror the phylogeny of Annelida as presently understood, and we found evidence of extensive Hr gene duplication and loss in annelids. Gene tree topology supported monophyly of cHrs and a myoHr clade that included nHrs sequences, indicating these designations are functional rather than evolutionary. CONCLUSIONS: The presence of several cHrs in early branching taxa suggests that a variety of Hrs were present in the common ancestor of extant annelids. Although our analysis was limited to expressed-coding regions, our findings demonstrate a greater diversity of Hrs among annelids than previously reported.

Copolymerization of recombinant Phascolopsis gouldii hemerythrin with human serum albumin for use in blood substitutes.

Hemerythrin is an oxygen-carrying protein found in marine invertebrates and may be a promising alternative to hemoglobin for use in blood substitutes, primarily due to its negligible peroxidative toxicity. Previous studies have shown that glutaraldehyde-induced copolymerization of hemoglobin with bovine serum albumin increases the half-life of the active oxy form of hemoglobin (i.e. decreases the auto-oxidation rate). Here, we describe a protocol for glutaraldehyde copolymerization of Hr with human serum albumin and the dioxygen-binding properties of the co-polymerized products. The copolymerization with HSA results in alteration of hemerythrin's dioxygen-binding properties in directions that may be favorable for use in blood substitutes.

Hemerythrin-related antimicrobial peptide, msHemerycin, purified from the body of the Lugworm, Marphysa sanguinea.

A ∼1.7 kDa antimicrobial peptide was purified from the acidified body extract of the Lugworm, Marphysa sanguinea, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide is composed of 14 amino acids with the N-terminal acetylation. Comparison of the identified amino acid sequences and molecular weight of this peptide with those of other known proteins or peptides revealed that this peptide had high identity to the N-terminus of hemerythrin of marine invertebrates and named the msHemerycin. The full-length hemerythrin cDNA of Lugworm was contained 1027-bp, including a 5'-untranslated region (UTR) of 60-bp, a 3'-UTR of 595-bp, and an open reading frame of 372-bp encoding 123 amino acids including the msHemerycin at the N-terminus. Tissue distribution of the msHemerycin mRNA suggests that it is constitutively expressed as a non-tissue-specific manner, however, a relatively higher expression level was observed in muscle (6.8-fold) and brain (6.3-fold), and the lowest level in digestive gland. The secondary structural prediction and homology modeling studies indicate that the msHemerycin might form an unordered structure and might act via unconventional mechanism. Our results suggest that the msHemerycin might be an innate immune component related to the host defenses in the Lugworm. This is the first report on the antimicrobial function of the peptide derived from the N-terminus of hemerythrin in the Lugworm, Marphysa sanguinea.

Molecular Evolution of the Oxygen-Binding Hemerythrin Domain.

BACKGROUND: The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins) are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes. RESULTS: Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid) cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role. CONCLUSIONS: Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later evolution of the oxygen-binding hemerythrin domain in both prokaryotes and eukaryotes led to a wide variety of functions, ranging from protection against oxidative damage in anaerobic and microaerophilic organisms, to oxygen supplying to particular enzymes and pathways in aerobic and facultative species.

De novo design of protein-protein interactions through modification of inter-molecular helix-helix interface residues.

For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy-to-use approach has also been reported that arranges leucines on a solvent-accessible region of an α-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an α-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysLARFH-IV-3L3R and the sulerythrin mutant 6L6D (KD=0.16 µM). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy-to-use approach to create de novo interfaces on the α-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures.

The bacteriohemerythrin from Methylococcus capsulatus (Bath): Crystal structures reveal that Leu114 regulates a water tunnel.

The bacteriohemerythrin (McHr) from Methylococcus capsulatus (Bath) is an oxygen carrier that serves as a transporter to deliver O2 from the cytosol of the bacterial cell body to the particulate methane monooxygenase residing in the intracytoplasmic membranes for methane oxidation. Here we report X-ray protein crystal structures of the recombinant wild type (WT) McHr and its L114A, L114Y and L114F mutants. The structure of the WT reveals a possible water tunnel in the McHr that might be linked to its faster autoxidation relative to hemerythrin in marine invertebrates. With Leu114 positioned at the end of this putative water tunnel, the hydrophobic side chain of this residue seems to play a prominent role in controlling the access of the water molecule required for autoxidation. This hypothesis is examined by comparing the autoxidation rates of the WT McHr with those of the L114A, L114Y and L114F mutants. The biochemical data are correlated with structural insights derived from the analysis of the putative water tunnels in the various McHr proteins provided by the X-ray structures.

Oxidative protection of hemoglobin and hemerythrin by cross-linking with a nonheme iron peroxidase: potentially improved oxygen carriers for use in blood substitutes.

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

F-box and leucine-rich repeat protein 5 (FBXL5): sensing intracellular iron and oxygen.

Though essential for many vital biological processes, excess iron results in the formation of damaging reactive oxygen species (ROS). Therefore, iron metabolism must be tightly regulated. F-box and leucine-rich repeat protein 5 (FBXL5), an E3 ubiquitin ligase subunit, regulates cellular and systemic iron homeostasis by facilitating iron regulatory protein 2 (IRP2) degradation. FBXL5 possesses an N-terminal hemerythrin (Hr)-like domain that mediates its own differential stability by switching between two different conformations to communicate cellular iron availability. In addition, the FBXL5-Hr domain also senses O2 availability, albeit by a distinct mechanism. Mice lacking FBXL5 fail to sense intracellular iron levels and die in utero due to iron overload and exposure to damaging levels of oxidative stress. By closely monitoring intracellular levels of iron and oxygen, FBLX5 prevents the formation of conditions that favor ROS formation. These findings suggest that FBXL5 is essential for the maintenance of iron homeostasis and is a key sensor of bioavailable iron. Here, we describe the iron and oxygen sensing mechanisms of the FBXL5 Hr-like domain and its role in mediating ROS biology.

H2O2-dependent substrate oxidation by an engineered diiron site in a bacterial hemerythrin.

The O2-binding carboxylate-bridged diiron site in DcrH-Hr was engineered in an effort to perform the H2O2-dependent oxidation of external substrates. A His residue was introduced near the diiron site in place of a conserved residue, Ile119. The I119H variant promotes the oxidation of guaiacol and 1,4-cyclohexadiene upon addition of H2O2.

[A hemerythrin-like protein MSMEG_3312 influences erythromycin resistance in mycobacteria].

OBJECTIVE: Reactive oxygen species are natural products of metabolism in aerobic organisms, which lead to oxidative damage, such as DNA mutation, protein inactivation and drug resistance. MSMEG_3312 was predicted as a hemerythrin-like protein, which can carry oxygen and reversibly bind to oxygen, thus it might play important roles in the process of oxygen metabolism. In this study, we explored the role of MSMEG_3312 in drug resistance. METHODS: On the basis of bioinformatics, we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical α-helix at secondary structure. To explore potential functions of MSMEG_3312, we constructed the msmeg_3312 knockout strain and compare the susceptibility to various drugs to its parent strain, mc2155. In addition, we also measured the promoter response when treatment of erythromycin. RESULTS: Genetic results showed that MSMEG_3312 is not necessary for M. smegmatis growth at 7H9 rich medium. The msmeg_3312 knockout strain showed increased erythromycin resistance. Moreover, the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis. However, there were no different MICs of other antibiotics, targets for protein synthesis inhibition, but not 50S subunit, such as tetracyclines, aminoglycosides and chloramphenicol. Moreover, we also showed that the promoter of msmeg_3312 responses to erythromycin. CONCLUSIONS: Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.

Nitrite activation to nitric oxide via one-fold protonation of iron(II)-O,O-nitrito complex: relevance to the nitrite reductase activity of deoxyhemoglobin and deoxyhemerythrin.

The reversible transformations [(Bim)3Fe(κ(2)-O2N)][BF4] (3) <-> [(Bim)3Fe(NO)(κ(1)-ONO)][BF4]2 (4) were demonstrated and characterized. Transformation of O,O-nitrito-containing complex 3 into [(Bim)3Fe(µ-O)(µ-OAc)Fe(Bim)3](3+) (5) along with the release of NO and H2O triggered by 1 equiv of AcOH implicates that nitrite-to-nitric oxide conversion occurs, in contrast to two protons needed to trigger nitrite reduction producing NO observed in the protonation of [Fe(II)-nitro] complexes.