Digital colloid-enhanced Raman spectroscopy by single-molecule counting.

Quantitative detection of various molecules at very low concentrations in complex mixtures has been the main objective in many fields of science and engineering, from the detection of cancer-causing mutagens and early disease markers to environmental pollutants and bioterror agents1-5. Moreover, technologies that can detect these analytes without external labels or modifications are extremely valuable and often preferred6. In this regard, surface-enhanced Raman spectroscopy can detect molecular species in complex mixtures on the basis only of their intrinsic and unique vibrational signatures7. However, the development of surface-enhanced Raman spectroscopy for this purpose has been challenging so far because of uncontrollable signal heterogeneity and poor reproducibility at low analyte concentrations8. Here, as a proof of concept, we show that, using digital (nano)colloid-enhanced Raman spectroscopy, reproducible quantification of a broad range of target molecules at very low concentrations can be routinely achieved with single-molecule counting, limited only by the Poisson noise of the measurement process. As metallic colloidal nanoparticles that enhance these vibrational signatures, including hydroxylamine-reduced-silver colloids, can be fabricated at large scale under routine conditions, we anticipate that digital (nano)colloid-enhanced Raman spectroscopy will become the technology of choice for the reliable and ultrasensitive detection of various analytes, including those of great importance for human health.

Inhibition of kynurenine production by N,O-substituted hydroxylamine derivatives.

The inhibition of kynurenine production is considered a promising target for cancer immunotherapy. In this study, an amino acid derivative, compound 1 was discovered using a cell-based assay with our screening library. Compound 1 suppressed kynurenine production without inhibiting indoleamine 2,3-dioxygenase 1 (IDO1) activity. The activity of 1 was derived from the inhibition of IDO1 by a metabolite of 1, O-benzylhydroxylamine (OBHA, 2a). A series of N-substituted 2a derivatives that exhibit potent activity in cell-based assays may represent effective prodrugs. Therefore, we synthesized and evaluated novel N,O-substituted hydroxylamine derivatives. The structure-activity relationships revealed that N,O-substituted hydroxylamine 2c inhibits kynurenine production in a cell-based assay. We conducted an in vivo experiment with 2c, although the effectiveness of O-substituted hydroxylamine derivatives in vivo has not been previously reported. The results indicate that N,O-substituted hydroxylamine derivatives are promising IDO1 inhibitors.

Amino acid and protein specificity of protein fatty acylation in C. elegans.

Protein lipidation plays critical roles in regulating protein function and localization. However, the chemical diversity and specificity of fatty acyl group utilization have not been investigated using untargeted approaches, and it is unclear to what extent structures and biosynthetic origins of S-acyl moieties differ from N- and O-fatty acylation. Here, we show that fatty acylation patterns in Caenorhabditis elegans differ markedly between different amino acid residues. Hydroxylamine capture revealed predominant cysteine S-acylation with 15-methylhexadecanoic acid (isoC17:0), a monomethyl branched-chain fatty acid (mmBCFA) derived from endogenous leucine catabolism. In contrast, enzymatic protein hydrolysis showed that N-terminal glycine was acylated almost exclusively with straight-chain myristic acid, whereas lysine was acylated preferentially with two different mmBCFAs and serine was acylated promiscuously with a broad range of fatty acids, including eicosapentaenoic acid. Global profiling of fatty acylated proteins using a set of click chemistry-capable alkyne probes for branched- and straight-chain fatty acids uncovered 1,013 S-acylated proteins and 510 hydroxylamine-resistant N- or O-acylated proteins. Subsets of S-acylated proteins were labeled almost exclusively by either a branched-chain or a straight-chain probe, demonstrating acylation specificity at the protein level. Acylation specificity was confirmed for selected examples, including the S-acyltransferase DHHC-10. Last, homology searches for the identified acylated proteins revealed a high degree of conservation of acylation site patterns across metazoa. Our results show that protein fatty acylation patterns integrate distinct branches of lipid metabolism in a residue- and protein-specific manner, providing a basis for mechanistic studies at both the amino acid and protein levels.

Discovery of a Hydroxylamine-Based Brain-Penetrant EGFR Inhibitor for Metastatic Non-Small-Cell Lung Cancer.

Metastases to the brain remain a significant problem in lung cancer, as treatment by most small-molecule targeted therapies is severely limited by efflux transporters at the blood-brain barrier (BBB). Here, we report the discovery of a selective, orally bioavailable, epidermal growth factor receptor (EGFR) inhibitor, 9, that exhibits high brain penetration and potent activity in osimertinib-resistant cell lines bearing L858R/C797S and exon19del/C797S EGFR resistance mutations. In vivo, 9 induced tumor regression in an intracranial patient-derived xenograft (PDX) murine model suggesting it as a potential lead for the treatment of localized and metastatic non-small-cell lung cancer (NSCLC) driven by activating mutant bearing EGFR. Overall, we demonstrate that an underrepresented functional group in medicinal chemistry, the trisubstituted hydroxylamine moiety, can be incorporated into a drug scaffold without the toxicity commonly surmised to accompany these units, all while maintaining potent biological activity and without the molecular weight creep common to drug optimization campaigns.

Electroanalytical overview: the sensing of hydroxylamine.

One of the principal raw ingredients used in the manufacturing of pharmaceuticals, nuclear fuel, and semiconductors is hydroxylamine, a mutagenic and carcinogenic substance, ranking high on the list of environmental contaminants. Electrochemical methods for monitoring hydroxylamine have the advantage of being portable, quick, affordable, simple, sensitive, and selective enough to maintain adequate constraints in contrast with conventional yet laboratory based quantification methods. This review outlines the most recent advancements in electroanalysis directed toward the sensing of hydroxylamine. Potential future advancements in this field are also offered, along with a discussion of method validation and the use of such devices in real samples for the determination of hydroxylamine.

Chemical Cyclic Amplification: Hydroxylamine Boosts the Fenton Reaction for Versatile and Scalable Biosensing.

Nucleic acid detection is undoubtedly one of the most important research fields to meet the medical needs of genetic disease diagnosis, cancer treatment, and infectious disease prevention. However, the practical detection methods based on biological amplification are complex and time-consuming and require highly trained operators. Herein, we report a simple, rapid, and sensitive method for the nucleic acid assay by fluorescence or naked eye using chemical cyclic amplification. The addition of hydroxylamine (HA) during the Fenton reaction can continuously generate hydroxyl radicals (•OH) via Fe3+/Fe2+ cycle, termed as "hydroxylamine boosts the Fenton reaction (Fenton-HA system)". Meanwhile, the reducing substances, such as terephthalic acid or o-phenylenediamine, react with •OH to generate oxidized substances that can be recognized by the naked eye or detected by fluorescence so as to realize the detection of Fe3+. The concentration of Fe3+ has a good linear relationship with fluorescence intensity in the range of 0.1 to 100 nM, and the limit of detection is calculated to be 0.03 nM (S/N = 3). Subsequently, Fe was introduced into the nucleic acid hybridization system after the Fe source was transformed into Fe3+, and the nucleic acids were indirectly determined by this method. This Fenton-HA system was used for sensing HIV-DNA and miRNA-21 to verify the validity of this method in nucleic acid detection. The detection limits were as low as 2.5 pM for HIV-DNA and 3 pM for miRNA-21. We believe that our work has unlocked an efficient signal amplification strategy, which is expected to develop a new generation of highly sensitive chemical biosensors.

Effects of hydroxylamine on treatment of anaerobic digestate of pig manure in partial nitrification-anaerobic ammonium oxidation.

Partial nitrification-anaerobic ammonium oxidation (PN-anammox) was started up within 40 days by bioaugmentation and aeration control, and its performance in the treatment of anaerobic digestate of pig manure (ADPM) was evaluated. Inhibitors in ADPM decreased the nitrogen removal rate (NRR) by 0.24 g N/L/d. The effect and mechanism of hydroxylamine (NH2OH) alleviation of PN-anammox inhibition during ADPM treatment were investigated. As an intermediate product of anammox and ammonia-oxidizing bacteria, NH2OH strengthened energy metabolism, improved the activity and abundance of functional bacteria, and eliminated miscellaneous bacteria, increasing the average NRR by 31%. However, the average nitrous oxide emission was increased by 10.1% via hydroxylamine oxidation. The results showed that synergy and competition among nitrogen-transforming microorganisms were crucial for NRR and that NH2OH played an essential role in maintaining efficient operation. This study lays a foundation for restoring PN-anammox for treating livestock wastewater.

Model simulation and mechanism of Fe(0/II/III) cycle activated persulfate degradation of methylparaben based on hydroxylamine enhanced nano-zero-valent iron.

The mechanism of Fe2+-activated peroxodisulfate (PDS) by hydroxylamine (HA) has been investigated, however, nano zero-valent iron-activated persulfate (nZVI/PDS) has a more optimal effect and needs further investigation. This study investigated the addition of HA to nZVI/PDS to improve Fe2+ regeneration and accelerate methylparaben (MP) degradation by Fe (0/II/III) cycle. After 60 min of reaction, the HA-enhanced nZVI/PDS (HA/nZVI/PDS) system afforded a 21% increase in MP degradation, reaching 93.26% (1 mM HA, 1 mM nZVI, and 2 mM PDS). nZVI/PDS system was a second-order reaction, but after adding HA, the reaction was more suitable for the first-order reaction. The addition of HA effectively promoted the reduction of Fe3+ to Fe2+ to improve the effect and reaction rate of PDS degradation of MP (k increased from 0.0127 min-1 to 0.0198 min-1) and broadened the reaction pH range. The results of various characterizations of nZVI before and after the reaction revealed that nZVI changed from a spherical structure to a bundle structure and was slightly oxidized. Changes in the Fe2+ and Fe3+ concentrations as well as in the pH of the reaction systems were monitored and the possible reactions of the HA/nZVI/PDS system were derived for the first time (knZVI/PDS<3.7 × 106 M-1 s-1, kFe3+/NH2O· >4.2 min-1). 12 potential compounds were investigated and MP breakdown pathways were speculated; hydroxylation was determined to be the most important pathway of degradation. And the HA/nZVI/PDS system had universal applicability.

Hydroxylamine-induced oxidation of ferrous nitrobindins.

Hemoglobin and myoglobin are generally taken as molecular models of all-α-helical heme-proteins. On the other hand, nitrophorins and nitrobindins (Nb), which are arranged in 8 and 10 ß-strands, respectively, represent the molecular models of all-ß-barrel heme-proteins. Here, kinetics of the hydroxylamine- (HA-) mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II), respectively), at pH 7.0 and 20.0 °C, are reported. Of note, HA displays antibacterial properties and is a good candidate for the treatment and/or prevention of reactive nitrogen species- (RNS-) linked aging-related pathologies, such as macular degeneration. Under anaerobic conditions, mixing the Mt-Nb(II), At-Nb(II), and Hs-Nb(II) solutions with the HA solutions brings about absorbance spectral changes reflecting the formation of the ferric derivative (i.e., Mt-Nb(III), At-Nb(III), and Hs-Nb(III), respectively). Values of the second order rate constant for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) are 1.1 × 104 M-1 s-1, 6.5 × 104 M-1 s-1, and 2.2 × 104 M-1 s-1, respectively. Moreover, the HA:Nb(II) stoichiometry is 1:2 as reported for ferrous deoxygenated and carbonylated all-α-helical heme-proteins. A comparative look of the HA reduction kinetics by several ferrous heme-proteins suggests that an important role might be played by residues (such as His or Tyr) in the proximity of the heme-Fe atom either coordinating it or not. In this respect, Nbs seem to exploit somewhat different structural aspects, indicating that redox mechanisms for the heme-Fe(II)-to-heme-Fe(III) conversion might differ between all-α-helical and all-ß-barrel heme-proteins.

Interactions of N-hydroxyamphetamine with an iron porphyrin: A unique intramolecular H-bond probed by DFT calculations.

Hydroxylamine (NH2OH) and its N-substituted derivatives (RNHOH) are important biological intermediates in the global N cycle. Heme plays a central role in the binding and activation of these hydroxylamines. We report the crystal structures of N-hydroxyamphetamine (AmphNHOH) in complex with Fe and Co heme models. We demonstrate a previously unrecognized internal H-bond interaction between a hydroxylamine RNHO-H group and a porphyrin N-atom. We utilize density functional theoretical (DFT) calculations to show that the conformations with the internal H-bond represent global minima along the potential energy surfaces for both the Fe and Co heme models. A natural bond orbital (NBO) analysis reveals a donor π (porN=C) to acceptor σ* (O-H) interaction of 3.04 kcal/mol for Fe, accounting for 11% of the total heme-AmphNHOH interaction energy. Our DFT calculations with the parent Fe-NH2OH suggests that the presence of internal H-bonds between hydroxylamine (R/H)NHOH moieties and heme N-atoms may be more common than previously recognized.

Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

[Precise identification of O-linked ß-N-acetylglucosamine peptides based on O-mesitylenesulfonylhydroxylamine elimination reaction].

O-linked ß-N-acetylglucosamine (O-GlcNAc), an important post-translational modification of proteins, plays an essential role in maintaining normal biological activities of organisms. Studies have shown that the disruption of O-GlcNAc homeostasis is closely associated with various human diseases. Therefore, large-scale enrichment and identification of O-GlcNAc proteins is important for exploring diagnostic biomarkers of clinical diseases. O-GlcNAcylation is substoichiometric, and its glycosidic bond is unstable; hence, the enrichment and identification of O-GlcNAc proteins remains a challenge. Recently, metabolic labeling technology with per-O-acetylated unnatural monosaccharides has been introduced to enrich O-GlcNAc proteins or peptides in cells. However, azidosugars can also react with the thiol group of cysteine to form cysteine thiol-azidosugar artificial modification as a byproduct in cell metabolism, which interferes with the identification of O-GlcNAc proteins or peptides. Therefore, the development of a methodology for the specific and complete removal of cysteine thiol-azidosugar artificial modification is necessary. O-Mesitylenesulfonylhydroxylamine (MSH) is an oxidizing and aminating reagent with great potential to oxidatively eliminate the cysteine thiol modification to dehydroalanine. Hence, it is worthwhile to probe whether MSH can eliminate cysteine thiol-azidosugar artificial modification. Two cysteine thiol-azidosugar artificial modifications were synthesized successfully by incubating thiol standard peptides with tetraacetylated N-azidoacetylgalactosamine (Ac4GalNAz) in sodium carbonate buffer (200 mmol/L, pH 10) at 37 ℃ for 90 min. Then, the reaction conditions for MSH oxidative elimination were optimized to completely remove cysteine thiol-azidosugar artificial modification. Sodium phosphate buffer (50 mmol/L, pH 8.0) was selected to protect the O-GlcNAc modification due to its mild nature. After extensive investigation, the optimized reaction conditions were established as 95 ℃ (reaction temperature) and 30 min (reaction time). Both cysteine thiol-azidosugar artificial modifications could be removed completely under these conditions. Furthermore, two azide-labeled O-GlcNAc (N3-O-GlcNAc) peptides were used to assess whether MSH destroyed the O-GlcNAc modification at the same time. The results showed that the two N3-O-GlcNAc peptides were stable after treatment with MSH at 95 ℃ for 30 min. In short, with excess MSH, the N3-O-GlcNAc peptides were stable, but the cysteine thiol-azidosugar artificial modification was exhausted in sodium phosphate buffer (50 mmol/L, pH 8.0) at 95 ℃ for 30 min. Moreover, both O-GlcNAc modification and cysteine thiol-azidosugar artificial modification exist in cell metabolism. This method can not only remove cysteine thiol-azidosugar artificial modification but also ensure the stability of O-GlcNAc modification. There is growing evidence that O-GlcNAcylation mainly occurs in proteins in the cytoplasm and nucleus, and that most O-GlcNAc proteins are involved in important biological signaling pathways. The nuclear and cytoplasmic proteins incubated with Ac4GalNAz in HeLa cells were selected as a model system. After the nuclear and cytoplasmic proteins were digested into peptides, MSH was applied to remove cysteine thiol-azidosugar modification. In addition, 51 peptides for the elimination of the cysteine thiol-azidosugar artificial modification were identified, indicating that MSH can remove the interference of cysteine thiol-azidosugar artificial modification in cell metabolism. Biotin probe and streptavidin dynabeads were subsequently used to label and enrich N3-O-GlcNAz peptides in cell metabolism. Finally, 157 O-GlcNAc peptides attributed to 130 proteins were identified. To better understand the functional roles of O-GlcNAc proteins, gene ontology analysis was performed. Cell component analysis showed that the identified O-GlcNAc proteins were mainly distributed inpostsynaptic density, cytoplasm, and condensed nuclear chromosome. The proteins were mostly involved in biological processes, including cell division, excitatory postsynaptic potential, and microtubule-based movement. The proteins responsible for transferase activity, transferring acyl groups, histone acetyltransferase activity, and microtubule binding were highly enriched, indicating that O-GlcNAc proteins play important roles in cells. In summary, this work developed an approach to enrich O-GlcNAc peptides precisely in metabolic labeling by removing cysteine thiol-azidosugar artificial modification with MSH. This methodology provides a new strategy for the application of metabolic labeling technology with unnatural monosaccharides in glycoproteomics analysis.

Non-Hydroxamate Zinc-Binding Groups as Warheads for Histone Deacetylases.

Histone deacetylases (HDACs) remove acetyl groups from acetylated lysine residues and have a large variety of substrates and interaction partners. Therefore, it is not surprising that HDACs are involved in many diseases. Most inhibitors of zinc-dependent HDACs (HDACis) including approved drugs contain a hydroxamate as a zinc-binding group (ZBG), which is by far the biggest contributor to affinity, while chemical variation of the residual molecule is exploited to create more or less selectivity against HDAC isozymes or other metalloproteins. Hydroxamates have a propensity for nonspecificity and have recently come under considerable suspicion because of potential mutagenicity. Therefore, there are significant concerns when applying hydroxamate-containing compounds as therapeutics in chronic diseases beyond oncology due to unwanted toxic side effects. In the last years, several alternative ZBGs have been developed, which can replace the critical hydroxamate group in HDACis, while preserving high potency. Moreover, these compounds can be developed into highly selective inhibitors. This review aims at providing an overview of the progress in the field of non-hydroxamic HDACis in the time period from 2015 to present. Formally, ZBGs are clustered according to their binding mode and structural similarity to provide qualitative assessments and predictions based on available structural information.

Activation of peroxymonosulfate by iron oxychloride with hydroxylamine for ciprofloxacin degradation and bacterial disinfection.

Iron oxychloride (FeOCl) is a known effective iron-based catalyst and has been used in advanced oxidation processes (AOPs). This study intends to achieve more facile free radicals generation from peroxymonosulfate (PMS) activation by exploring the Fe(III)/Fe(II) cycle of FeOCl in the presence of hydroxylamine (HA). With 0.2 g/L FeOCl, 1.5 mM PMS, and 1 mM HA, the PMS/FeOCl/HA system could effectively achieve 98.88% of the oxidative degradation of 5 mg/L ciprofloxacin (CIP) in 15 min and quickly inactivate 99.99% of E. coli (108 CFU/mL) in 5 min at near-neutral pH. HA played an important role in promoting the Fe(III)/Fe(II) cycle, thereby greatly improving the oxidation activity of the system. The reactive oxygen species (ROS) such as HO, SO4- and O2- were identified as the dominated free radicals produced in the system. The intermediate products of CIP detected by liquid chromatograph-mass spectrometer (LC-MS) and three possible degradation pathways of CIP were proposed. The presence of common anions in the PMS/FeOCl/HA system, including HCO3-, Cl-, SO42-, and NO3-, enhanced the degradation efficiency of CIP to varying degrees at the concentrations of 10 mM. Moreover, FeOCl maintained a high degradation capability for CIP after several recycles. This work offers a new promising means of catalyzing the PMS-based AOPs in the degradation of refractory organics.

Complexes of Formaldehyde and α-Dicarbonyls with Hydroxylamine: FTIR Matrix Isolation and Theoretical Study.

The interactions of formaldehyde (FA), glyoxal (Gly) and methylglyoxal (MGly) with hydroxylamine (HA) isolated in solid argon and nitrogen were studied using FTIR spectroscopy and ab initio methods. The spectra analysis indicates the formation of two types of hydrogen-bonded complexes between carbonyl and hydroxylamine in the studied matrices. The cyclic planar complexes are stabilized by O-H⋯O(C), and C-H⋯N interactions and the nonplanar complexes are stabilized by O-H⋯O(C) bond. Formaldehyde was found to form with hydroxylamine, the cyclic planar complex and methylglyoxal, the nonplanar one in both argon and nitrogen matrices. In turn, glyoxal forms with hydroxylamine the most stable nonplanar complex in solid argon, whereas in solid nitrogen, both types of the complex are formed.

The Role of Hydrogen Sulfide in the Development of Tolerance and Dependence to Morphine in Mice.

OBJECTIVE: Hydrogen sulfide is an endogenous gaseous mediator that has been indicated to have a role in pain mechanisms. In this study, we aimed to detect brain and spinal cord hydrogen sulfide levels during different phases of tolerance and dependence to morphine and to determine the effects of inhibition of endogenous hydrogen sulfide production on the development of tolerance and dependence. METHODS: Morphine tolerance and dependence was developed by subcutaneous injection of morphine (10 mg/kg) twice daily for 12 days. Physical dependence was determined by counting the jumps for 20 min, which is a withdrawal symptom occurring after a single dose of naloxone (5 mg/kg) administered intraperitoneally (i.p.). Propargylglycine (30 mg/kg, i.p.), a cystathionine-γ-lyase inhibitor, and hydroxylamine (12.5 mg/kg, i.p.), a cystathionine-ß-synthase inhibitor, were used as hydrogen sulfide synthase inhibitors. The tail-flick and hot-plate tests were used to determine the loss of antinociceptive effects of morphine and development of tolerance. RESULTS: It was found that chronic and acute uses of both propargylglycine and hydroxylamine prevented the development of tolerance to morphine, whereas they had no effect on morphine dependence. Chronic and acute administrations of hydrogen sulfide synthase inhibitors did not exert any difference in hydrogen sulfide levels in brain and spinal cords of both morphine-tolerant and -dependent animals. CONCLUSION: It has been concluded that hydrogen sulfide synthase inhibitors may have utility in preventing morphine tolerance.

Heme P460: A (Cross) Link to Nitric Oxide.

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2-) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2-. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c-heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea, which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH2OH and were both originally reported to produce NO2-. Each can inspire means to effect controlled release of chemical energy.Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2- was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O2, thus producing NO2- abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2- is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2- enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen "defixation".Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH2OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NH2OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-links. The heme-Lys cross-link of cyt P460 enforces the relative position of the cofactor and second-sphere residues. Moreover, the cross-link prevents the dissociation of the axial histidine residue, which stops catalysis, emphasizing the importance of this unique post-translational modification.

Hydroxylamine Complexes of Cytochrome c': Influence of Heme Iron Redox State on Kinetic and Spectroscopic Properties.

Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme-HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochrome c nitrite reductase. Despite the importance of heme-HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme-HA affinity, kinetics, and spectroscopy. Using stopped-flow UV-vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant of Alcaligenes xylosoxidans cytochrome c'-α (L16G AxCP-α), a pentacoordinate c-type cytochrome that we show binds HA in its Fe(III) (Kd ∼ 2.5 mM) and Fe(II) (Kd = 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lower koff value (0.0994 s-1 vs 11 s-1), whereas kon values for Fe(II) (2880 M-1 s-1) and Fe(III) (4300 M-1 s-1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N-O stretching vibration of the NH2OH ligand, ν(N-O) = 906 cm-1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme-HA reaction mechanisms.

Design and Scalable Synthesis of N-Alkylhydroxylamine Reagents for the Direct Iron-Catalyzed Installation of Medicinally Relevant Amines*.

Secondary and tertiary alkylamines are privileged substance classes that are often found in pharmaceuticals and other biologically active small molecules. Herein, we report their direct synthesis from alkenes through an aminative difunctionalization reaction enabled by iron catalysis. A family of ten novel hydroxylamine-derived aminating reagents were designed for the installation of several medicinally relevant amine groups, such as methylamine, morpholine and piperazine, through the aminochlorination of alkenes. The method has excellent functional group tolerance and a broad scope of alkenes was converted to the corresponding products, including several drug-like molecules. Besides aminochlorination, the installation of other functionalities through aminoazidation, aminohydroxylation and even intramolecular carboamination reactions, was demonstrated, further highlighting the broad potential of these new reagents for the discovery of novel amination reactions.