Mechanism and Kinetics of Inducible Nitric Oxide Synthase Auto-<i>S</i>-nitrosation and Inactivation

Nitric oxide (NO), the product of the nitric oxide synthase (NOS) reaction, was previously shown to result in <i>S</i>-nitrosation of the NOS Zn²⁺-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOS <i>S</i>-nitrosation, we determined the inactivation time scale of the inducible NOS isoform (iNOS) and found it directly correlates with an increase in the level of iNOS <i>S</i>-nitrosation. A kinetic model of NOS inactivation in which arginine is treated as a suicide substrate was developed. In this model, NO synthesized at the heme cofactor is partitioned between release into solution (NO release pathway) and NOS <i>S-</i>nitrosation followed by NOS inactivation (inactivation pathway). Experimentally determined progress curves of NO formation were fit to the model. The NO release pathway was perturbed through addition of the NO traps oxymyoglobin (MbO₂) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ∼100 at 4 μM MbO₂ and ∼22000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn²⁺-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agent GSH or TCEP resulted in a concentration-dependent decrease in the level of iNOS <i>S</i>-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparent <i>K</i>_m value of 13 mM. NOS turnover that leads to NOS <i>S</i>-nitrosation might be a mechanism for controlling NOS activity, and NOS <i>S-</i>nitrosation could play a role in the physiological generation of nitrosothiols

Nitric Oxide-Induced Conformational Changes Govern H‑NOX and Histidine Kinase Interaction and Regulation in <i>Shewanella oneidensis</i>

Nitric oxide (NO) is implicated in biofilm regulation in several bacterial families via heme-nitric oxide/oxygen binding (H-NOX) protein signaling. Shewanella oneidensis H-NOX (<i>So</i> H-NOX) is associated with a histidine kinase (<i>So</i> HnoK) encoded on the same operon, and together they form a multicomponent signaling network whereby the NO-bound state of <i>So</i> H-NOX inhibits <i>So</i> HnoK autophosphorylation activity, affecting the phosphorylation state of three response regulators. Although the conformational changes of <i>So</i> H-NOX upon NO binding have been structurally characterized, the mechanism of HnoK inhibition by NO-bound <i>So</i> H-NOX remains unclear. In the present study, the molecular details of <i>So</i> H-NOX and <i>So</i> HnoK interaction and regulation are characterized. The N-terminal domain in <i>So</i> HnoK was determined to be the site of H-NOX interaction, and the binding interface on <i>So</i> H-NOX was identified using a combination of hydrogen–deuterium exchange mass spectrometry and surface-scanning mutagenesis. Binding kinetics measurements and analytical gel filtration revealed that NO-bound <i>So</i> H-NOX has a tighter affinity for <i>So</i> HnoK compared that of H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with binding-deficient H-NOX mutants further indicate that while formation of the H-NOX-HnoK complex is required for HnoK to be catalytically active, H-NOX conformational changes upon NO-binding are necessary for HnoK inhibition

Mapping the H‑NOX/HK Binding Interface in <i>Vibrio cholerae</i> by Hydrogen/Deuterium Exchange Mass Spectrometry

Heme-nitric oxide/oxygen binding (H-NOX) proteins are a group of hemoproteins that bind diatomic gas ligands such as nitric oxide (NO) and oxygen (O₂). H-NOX proteins typically regulate histidine kinases (HK) located within the same operon. It has been reported that NO-bound H-NOXs inhibit cognate histidine kinase autophosphorylation in bacterial H-NOX/HK complexes; however, a detailed mechanism of NO-mediated regulation of the H-NOX/HK activity remains unknown. In this study, the binding interface of <i>Vibrio cholerae</i> (<i>Vc</i>) H-NOX/HK complex was characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and further validated by mutagenesis, leading to a new model for NO-dependent kinase inhibition. A conformational change in <i>Vc</i> H-NOX introduced by NO generates a new kinase-binding interface, thus locking the kinase in an inhibitory conformation

Native Alanine Substitution in the Glycine Hinge Modulates Conformational Flexibility of Heme Nitric Oxide/Oxygen (H-NOX) Sensing Proteins

Heme nitric oxide/oxygen sensing (H-NOX) domains are direct NO sensors that regulate a variety of biological functions in both bacteria and eukaryotes. Previous work on H-NOX proteins has shown that upon NO binding, a conformational change occurs along two glycine residues on adjacent helices (termed the glycine hinge). Despite the apparent importance of the glycine hinge, it is not fully conserved in all H-NOX domains. Several H-NOX sensors from the family Flavobacteriaceae contain a native alanine substitution in one of the hinge residues. In this work, the effect of the increased steric bulk within the Ala-Gly hinge on H-NOX function was investigated. The hinge in <i>Kordia algicida</i> OT-1 (<i>Ka</i> H-NOX) is composed of A71 and G145. Ligand-binding properties and signaling function for this H-NOX were characterized. The variant A71G was designed to convert the hinge region of <i>Ka</i> H-NOX to the typical Gly-Gly motif. In activity assays with its cognate histidine kinase (HnoK), the wild type displayed increased signal specificity compared to A71G. Increasing titrations of unliganded A71G gradually inhibits HnoK autophosphorylation, while increasing titrations of unliganded wild type H-NOX does not inhibit HnoK. Crystal structures of both wild type and A71G <i>Ka</i> H-NOX were solved to 1.9 and 1.6 Å, respectively. Regions of H-NOX domains previously identified as involved in protein–protein interactions with HnoK display significantly higher b-factors in A71G compared to wild-type H-NOX. Both biochemical and structural data indicate that the hinge region controls overall conformational flexibility of the H-NOX, affecting NO complex formation and regulation of its HnoK

Porphyrin-Substituted H‑NOX Proteins as High-Relaxivity MRI Contrast Agents

Heme proteins are exquisitely tuned to carry out diverse biological functions while employing identical heme cofactors. Although heme protein properties are often altered through modification of the protein scaffold, protein function can be greatly expanded and diversified through replacement of the native heme with an unnatural porphyrin of interest. Thus, porphyrin substitution in proteins affords new opportunities to rationally tailor heme protein chemical properties for new biological applications. Here, a highly thermally stable Heme Nitric oxide/OXygen binding (H-NOX) protein is evaluated as a magnetic resonance imaging (MRI) contrast agent. <i>T</i>₁ and <i>T</i>₂ relaxivities measured for the H-NOX protein containing its native heme are compared to the protein substituted with unnatural manganese­(II/III) and gadolinium­(III) porphyrins. H-NOX proteins are found to provide unique porphyrin coordination environments and have enhanced relaxivities compared to commercial small-molecule agents. Porphyrin substitution is a promising strategy to encapsulate MRI-active metals in heme protein scaffolds for future imaging applications

Determinants of Regioselective Hydroxylation in the Fungal Polysaccharide Monooxygenases

The ubiquitous fungal poly­saccharide mono­oxy­genases (PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are structurally related but have significant variation in sequence. A heterologous expression method in <i>Neurospora crassa</i> was developed as a step toward connecting regioselectivity of the chemistry to PMO phylogeny. Activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal PMOs fall into three major groups with distinctive active site surface features. PMO1s and PMO2s hydroxylate glycosidic positions C1 and C4, respectively. PMO3s hydroxylate both C1 and C4. A subgroup of PMO3s (PMO3*) hydroxylate C1. Mutagenesis studies showed that an extra subdomain of about 12 amino acids contribute to C4 oxidation in the PMO3 family

Toward ‘Omic Scale Metabolite Profiling: A Dual Separation–Mass Spectrometry Approach for Coverage of Lipid and Central Carbon Metabolism

Although the objective of any ‘omic science is broad measurement of its constituents, such coverage has been challenging in metabolomics because the metabolome is comprised of a chemically diverse set of small molecules with variable physical properties. While extensive studies have been performed to identify metabolite isolation and separation methods, these strategies introduce bias toward lipophilic or water-soluble metabolites depending on whether reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) is used, respectively. Here we extend our consideration of metabolome isolation and separation procedures to integrate RPLC/MS and HILIC/MS profiling. An aminopropyl-based HILIC/MS method was optimized on the basis of mobile-phase additives and pH, followed by evaluation of reproducibility. When applied to the untargeted study of perturbed bacterial metabolomes, the HILIC method enabled the accurate assessment of key, dysregulated metabolites in central carbon pathways (e.g., amino acids, organic acids, phosphorylated sugars, energy currency metabolites), which could not be retained by RPLC. To demonstrate the value of the integrative approach, bacterial cells, human plasma, and cancer cells were analyzed by combined RPLC/HILIC separation coupled to ESI positive/negative MS detection. The combined approach resulted in the observation of metabolites associated with lipid and central carbon metabolism from a single biological extract, using 80% organic solvent (ACN:MeOH:H₂O 2:2:1). It enabled the detection of more than 30,000 features from each sample type, with the highest number of uniquely detected features by RPLC in ESI positive mode and by HILIC in ESI negative mode. Therefore, we conclude that when time and sample are limited, the maximum amount of biological information related to lipid and central carbon metabolism can be acquired by combining RPLC ESI positive and HILIC ESI negative mode analysis