Alternative Metal with an Alternative Mechanism for Metalloporphyrin-Enabled NO Reduction to NO: A Combined Computational and Experimental Investigation of NO Reduction by Cr Porphyrin with Lewis Acid.

Nitric oxide (NO) is an important molecule that regulates many physiological processes in humans and plants and contributes to the formation of greenhouse gases. Bacterial NO reductases utilize a di-Fe hemenonheme active site to couple two NOs to generate nitrous oxide (NO) via a two-electron mechanism. Here, we report a previously unexplored Cr porphyrin NO complex with a Lewis acid (LA) BF for the NO reduction reaction.

One-Electron NO to NO Pathways via Heme Models and Lewis Acid: Metal Effects and Differences from the Enzymatic Reaction.

Some pathogens use heme-containing nitric oxide reductases (NORs) to reduce NO to NO as their defense mechanism to detoxify NO and reduce nitrosative stress. This reduction is also significant in the global N cycle. Our previous experimental work showed that Fe and Co porphyrin NO complexes can couple with external NO to form NO when activated by the Lewis acid BF.

Vibrational Coupling Infrared Nanocrystallography.

Coupling between molecular vibrations leads to collective vibrational states with spectral features sensitive to local molecular order. This provides spectroscopic access to the low-frequency intermolecular energy landscape. In its nanospectroscopic implementation, this technique of vibrational coupling nanocrystallography (VCNC) offers information on molecular disorder and domain formation with nanometer spatial resolution.

Interactions of metronidazole and chloramphenicol with myoglobin: Crystal structure of a Mb-acetamide product.

Nitroorganics present a general concern for a safe environment due to their health hazards. However, some nitroorganics such as metronidazole (Mtz) and chloramphenicol (CAM) also possess medicinal value. Mtz and CAM can undergo reductive bioactivation presumably via their nitroso derivatives.

Interactions of arylhydroxylamines and alkylaldoximes with a rhodium porphyrin.

Heme enzymes are involved in the binding and metabolism of hydroxylamine (RNHOH) and aldoxime (RCH=NOH) compounds (R = H, alkyl, aryl). We report the synthesis and X-ray crystal structure of a metalloporphyrin in complex with an arylhydroxylamine, namely that of (TPP)Rh(PhNHOH)(CHCl) (TPP = tetraphenylpophryinato dianion). The crystal structure reveals, in addition to N-binding of PhNHOH to Rh, the presence of an intramolecular H-bond between the hydroxylamine -OH proton and a porphyrin N-atom.

Crystal structural investigations of heme protein derivatives resulting from reactions of aryl- and alkylhydroxylamines with human hemoglobin.

Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV-vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me, t-Bu) react with human ferric Hb.

Insights into Nitrosoalkane Binding to Myoglobin Provided by Crystallography of Wild-Type and Distal Pocket Mutant Derivatives.

Nitrosoalkanes (R-N═O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH) drugs or from the reduction of nitroorganics (RNO). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe-RNO moieties remains limited.

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

Hydroxylamine (NHOH) 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.

Insights into the Observed -Bond Length Variations upon NO Binding to Ferric and Ferrous Porphyrins with Neutral Axial Ligands.

NO is well-known for its effect. NO binding to ferrous hemes of the form (por)Fe(L) (L = neutral N-based ligand) to give the {FeNO} (por)Fe(NO)(L) product results in a lengthening of the axial Fe-L bond. In contrast, NO binding to the ferric center in [(por)Fe(L)] to give the {FeNO} [(por)Fe(NO)(L)] product results in a shortening of the Fe-L bond.

Insight into the preferential N-binding O-binding of nitrosoarenes to ferrous and ferric heme centers.

Nitrosoarenes (ArNOs) are toxic metabolic intermediates that bind to heme proteins to inhibit their functions. Although much of their biological functions involve coordination to the Fe centers of hemes, the factors that determine N-binding or O-binding of these ArNOs have not been determined. We utilize X-ray crystallography and density functional theory (DFT) analyses of new representative ferrous and ferric ArNO compounds to provide the first theoretical insight into preferential N-binding versus O-binding of ArNOs to hemes.

The nitrosoamphetamine metabolite is accommodated in the active site of human hemoglobin: Spectroscopy and crystal structure.

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group.

Not Limited to Iron: A Cobalt Heme-NO Model Facilitates N-N Coupling with External NO in the Presence of a Lewis Acid to Generate N O.

Some bacterial heme proteins catalyze the coupling of two NO molecules to generate N O. We previously reported that a heme Fe-NO model engages in this N-N bond-forming reaction with NO. We now demonstrate that (OEP)Co (NO) similarly reacts with 1 equiv of NO in the presence of the Lewis acids BX (X=F, C F ) to generate N O.

Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen.

The R20291 genome encodes 57 response regulator proteins that, as part of two-component signaling pathways, regulate adaptation to environmental conditions. Genomic and transcriptomic studies in have been limited, due to technical challenges, to the analysis of either high-throughput screens or high-priority targets, such as primary regulators of toxins or spore biology. We present the use of several technically accessible and generally applicable techniques to elucidate the putative regulatory targets of a response regulator, RR_1586, involved in sporulation of the hypervirulent strain R20291.

Nitrosyl Myoglobins and Their Nitrite Precursors: Crystal Structural and Quantum Mechanics and Molecular Mechanics Theoretical Investigations of Preferred Fe -NO Ligand Orientations in Myoglobin Distal Pockets.

The globular dioxygen binding heme protein myoglobin (Mb) is present in several species. Its interactions with the simple nitrogen oxides, namely, nitric oxide (NO) and nitrite, have been known for decades, but the physiological relevance has only recently become more fully appreciated. We previously reported the O-nitrito mode of binding of nitrite to ferric horse heart wild-type (wt) Mb and human hemoglobin.

Lewis Acid Activation of the Ferrous Heme-NO Fragment toward the N-N Coupling Reaction with NO To Generate NO.

Bacterial NO reductase (bacNOR) enzymes utilize a heme/non-heme active site to couple two NO molecules to NO. We show that BF coordination to the nitrosyl O-atom in (OEP)Fe(NO) activates it toward N-N bond formation with NO to generate NO. N-isotopic labeling reveals a reversible nitrosyl exchange reaction and follow-up N-O bond cleavage in the NO formation step.

Carbon-Nitrogen and Nitrogen-Nitrogen Bond Formation from Nucleophilic Attack at Coordinated Nitrosyls in Fe and Ru Heme Models.

The conversion of inorganic NO species to organo-N compounds is an important component of the global N-cycle. Reaction of a C-based nucleophile, namely the phenyl anion, with the ferric heme nitrosyl [(OEP)Fe(NO)(5-MeIm)] generates a mixture of the C-nitroso derivative (OEP)Fe(PhNO)(5-MeIm) and (OEP)Fe(Ph). The related reaction with [(OEP)Ru(NO)(5-MeIm)] generates the (OEP)Ru(PhNO)(5-MeIm) product.

Nitrosoamphetamine binding to myoglobin and hemoglobin: Crystal structure of the H64A myoglobin-nitrosoamphetamine adduct.

N-hydroxyamphetamine (AmphNHOH) is an oxidative metabolite of amphetamine and methamphetamine. It is known to form inhibitory complexes upon binding to heme proteins. However, its interactions with myoglobin (Mb) and hemoglobin (Hb) have not been reported.

Nitric oxide coupling to generate NO promoted by a single-heme system as examined by density functional theory.

Bacteria utilize a heme/non-heme enzyme system to detoxify nitric oxide (NO) to NO. In order to probe the capacity of a single-heme system to mediate this NO-to-NO transformation, various scenarios for addition of electrons, protons, and a second NO molecule to a heme nitrosyl to generate NO were explored by density functional theory calculations. We describe, utilizing this single-heme system, several stepwise intermediates along pathways that enable the critical N-N bond formation step yielding the desired Fe-NO product.

Crystal structures of two nitroreductases from hypervirulent Clostridium difficile and functionally related interactions with the antibiotic metronidazole.

Nitroreductases (NRs) are flavin mononucleotide (FMN)-dependent enzymes that catalyze the biotransformation of organic nitro compounds (RNO; R = alkyl, aryl) to the nitroso RN=O, hydroxylamino RNHOH, or amine RNH derivatives. Metronidazole (Mtz) is a nitro-containing antibiotic that is commonly prescribed for lower-gut infections caused by the anaerobic bacterium Clostridium difficile. C.

Crystal structure and DNA binding activity of a PadR family transcription regulator from hypervirulent Clostridium difficile R20291.

Background: Clostridium difficile is a spore-forming obligate anaerobe that can remain viable for extended periods, even in the presence of antibiotics, which contributes to the persistence of this bacterium as a human pathogen during host-to-host transmission and in hospital environments. We examined the structure and function of a gene product with the locus tag CDR20291_0991 (cdPadR1) as part of our broader goal aimed at elucidating transcription regulatory mechanisms involved in virulence and antibiotic resistance of the recently emergent hypervirulent C. difficile strain R20291.

Organometallic myoglobins: Formation of Fe-carbon bonds and distal pocket effects on aryl ligand conformations.

Bioorganometallic Fe-C bonds are biologically relevant species that may result from the metabolism of natural or synthetic hydrazines. The molecular structures of four new sperm whale mutant myoglobin derivatives with Fe-aryl moieties, namely H64A-tolyl-m, H64A-chlorophenyl-p, H64Q-tolyl-m, and H64Q-chlorophenyl-p, have been determined at 1.7-1.

Hydride Attack on a Coordinated Ferric Nitrosyl: Experimental and DFT Evidence for the Formation of a Heme Model-HNO Derivative.

Heme-HNO species are crucial intermediates in several biological processes. To date, no well-defined Fe heme-HNO model compounds have been reported. Hydride attack on the cationic ferric [(OEP)Fe(NO)(5-MeIm)]OTf (OEP = octaethylporphyrinato dianion) generates an Fe-HNO product that has been characterized by IR and (1)H NMR spectroscopy.

Six-coordinate ferric porphyrins containing bidentate N-t-butyl-N-nitrosohydroxylaminato ligands: structure, magnetism, IR spectroelectrochemisty, and reactivity.

NONOates (diazeniumdiolates) containing the [X{N2O2}](-) functional group are frequently employed as nitric oxide (NO) donors in biology, and some NONOates have been shown to bind to metalloenzymes. We report the preparation, crystal structures, detailed magnetic behavior, redox properties, and reactivities of the first isolable alkyl C-NONOate complexes of heme models, namely (OEP)Fe(η(2)-ON(t-Bu)NO) (1) and (TPP)Fe(η(2)-ON(t-Bu)NO) (2) (OEP = octaethylporphyrinato dianion, TPP = tetraphenylporphyrinato dianion). The compounds display the unusual NONOate O,O-bidentate binding mode for porphyrins, resulting in significant apical Fe displacements (+0.