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. However, deriving local molecular order relies on prior knowledge of the transition dipole magnitude and crystal structure of the underlying ordered phase. Here we develop a quantitative model for VCNC by relating nano-FTIR collective vibrational spectra to the molecular crystal structure from X-ray crystallography. We experimentally validate our approach at the example of a metal organic porphyrin complex with a carbonyl ligand as the probe vibration. This framework establishes VCNC as a powerful tool for measuring low-energy molecular interactions, wave function delocalization, nanoscale disorder, and domain formation in a wide range of molecular systems.

One-Electron NO to N2O 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 N2O 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 N2O when activated by the Lewis acid BF3. A key difference from conventional two-electron enzymatic reaction is that one electron is sufficient. However, a complete understanding of the entire reaction pathways and the more favorable reactivity for Fe remains unknown. Here, we present a quantum chemical study to provide such information. Our results confirmed Fe's higher experimental reactivity, showing advantages in all steps of the reaction pathway: easier metal oxidation for NO reduction and N-O cleavage as well as a larger size to expedite the N/O coordination mode transition. The Co system, with a similar product energy as the enzyme, shows potential for further development in catalytic NO coupling. This work also offers the first evidence that this new one-electron NO reduction is both kinetically competitive and thermodynamically more favorable than the native pathway, supporting future initiatives in optimizing NO reduction agents in biology, environment, and industry.

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 (RNH2) drugs or from the reduction of nitroorganics (RNO2). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe-RNO moieties remains limited. We report the preparation of ferrous wild-type and H64A sw MbII-RNO derivatives (λmax 424 nm; R = Me, Et, Pr, iPr) from the reactions of MbIII-H2O with dithionite and nitroalkanes. The apparent extent of formation of the wt Mb derivatives followed the order MeNO > EtNO > PrNO > iPrNO, whereas the order was the opposite for the H64A derivatives. Ferricyanide oxidation of the MbII-RNO derivatives resulted in the formation of the ferric MbIII-H2O precursors with loss of the RNO ligands. X-ray crystal structures of the wt MbII-RNO derivatives at 1.76-2.0 Å resoln. revealed N-binding of RNO to Fe and the presence of H-bonding interactions between the nitroso O-atoms and distal pocket His64. The nitroso O-atoms pointed in the general direction of the protein exterior, and the hydrophobic R groups pointed toward the protein interior. X-ray crystal structures for the H64A mutant derivatives were determined at 1.74-1.80 Å resoln. An analysis of the distal pocket amino acid surface landscape provided an explanation for the differences in ligand orientations adopted by the EtNO and PrNO ligands in their wt and H64A structures. Our results provide a good baseline for the structural analysis of RNO binding to heme proteins possessing small distal pockets.

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 N2 O.

Some bacterial heme proteins catalyze the coupling of two NO molecules to generate N2 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)CoII (NO) similarly reacts with 1 equiv of NO in the presence of the Lewis acids BX3 (X=F, C6 F5 ) to generate N2 O. DFT calculations support retention of the CoII oxidation state for the experimentally observed adduct (OEP)CoII (NO⋅BF3 ), the presumed hyponitrite intermediate (P.+ )CoII (ONNO⋅BF3 ), and the porphyrin π-radical cation by-product of this reaction, and that the π-radical cation formation likely occurs at the hyponitrite stage. In contrast, the Fe analogue undergoes a ferrous-to-ferric oxidation state conversion during this reaction. Our work shows that cobalt hemes are chemically competent to engage in the NO-to-N2 O conversion reaction.

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

The Clostridioides difficile 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 C. difficile 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 C. difficile strain R20291. A DNA-binding specificity motif for RR_1586 was determined using a bacterial one-hybrid assay originally developed for Drosophila transcription factors. Comparative bioinformatics approaches identified and in vitro experiments confirmed RR_1586 binding sites upstream of putative target genes, including those that encode phosphate ion transporters, spermidine/putrescine biosynthesis and transport pathways, ABC type transport systems, known regulators of sporulation, and genes encoding spore structural proteins. Representative examples of these regulatory interactions have been tested and confirmed in Escherichia coli-based reporter assays. Finally, evidence of possible regulatory mechanisms is also presented. A working model includes self-regulation by RR_1586 and phosphorylation-dependent and -independent DNA binding at low- and high-fidelity binding sites, respectively. Broad application of this and similar approaches is anticipated to be an important catalyst for the study of gene regulation by two-component systems from pathogenic or technically challenging bacteria.IMPORTANCEClostridioides difficile spores survive under harsh conditions and can germinate into actively dividing cells capable of causing disease. An understanding of the regulatory networks controlling sporulation and germination in C. difficile could be exploited for therapeutic advantage. However, such studies are hindered by the challenges of working with an anaerobic pathogen recalcitrant to genetic manipulation. Although two-component response regulators can be identified from genetic sequences, identification of their downstream regulatory networks requires further development. This work integrates experimental and bioinformatic approaches, which provide practical advantages over traditional transcriptomic analyses, to identify the putative regulon of the C. difficile response regulator RR_1586 by first screening for protein-DNA interactions in E. coli and then predicting regulatory outputs in C. difficile.

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) MbIII and human hemoglobin. We have expanded on this work and report the interactions of nitrite with wt sperm whale (sw) MbIII and its H64A, H64Q, and V68A/I107Y mutants whose dissociation constants increase in the following order: H64Q < wt < V68A/I107Y < H64A. We also report their X-ray crystal structures that reveal the O-nitrito mode of binding of nitrite to these derivatives. The MbII-mediated reductions of nitrite to NO and structural data for the wt and mutant MbII-NOs are described. We show that their FeNO orientations vary with distal pocket identity, with the FeNO moieties pointing toward the hydrophobic interiors when the His64 residue is present but toward the hydrophilic exterior when this His64 residue is absent in this set of mutants. This correlates with the nature of H-bonding to the bound NO ligand (nitrosyl O vs N atom). Quantum mechanics and hybrid quantum mechanics and molecular mechanics calculations help elucidate the origin of the experimentally preferred NO orientations. In a few cases, the calculations reproduce the experimentally observed orientations only when the whole protein is taken into consideration.

Lewis Acid Activation of the Ferrous Heme-NO Fragment toward the N-N Coupling Reaction with NO To Generate N2O.

Bacterial NO reductase (bacNOR) enzymes utilize a heme/non-heme active site to couple two NO molecules to N2O. We show that BF3 coordination to the nitrosyl O-atom in (OEP)Fe(NO) activates it toward N-N bond formation with NO to generate N2O. 15N-isotopic labeling reveals a reversible nitrosyl exchange reaction and follow-up N-O bond cleavage in the N2O formation step. Other Lewis acids (B(C6F5)3 and K+) also promote the NO coupling reaction with (OEP)Fe(NO). These results, complemented by DFT calculations, provide experimental support for the cis: b3 pathway in bacNOR.

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

The conversion of inorganic NOx 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. Reactions with the N-based nucleophile diethylamide results in the formation of free diethylnitrosamine, whereas the reaction with azide results in N2O formation; these products derive from attack of the nucleophiles on the bound NO groups. These results provide the first demonstrations of C-N and N-N bond formation from attack of C-based and N-based nucleophiles on synthetic ferric-NO hemes.

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. We demonstrate that the reactions of AmphNHOH with ferric Mb and Hb generate the respective heme-nitrosoamphetamine derivatives characterized by UV-vis spectroscopy. We have determined the X-ray crystal structure of the H64A Mb-nitrosoamphetamine complex to 1.73 Å resolution. The structure reveals the N-binding of the nitroso-d-amphetamine isomer, with no significant H-bonding interactions between the ligand and the distal pocket amino acid residues.

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. Results of DFT calculations reveal a direct attack of the hydride on the N atom of the coordinated ferric nitrosyl.

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. cdPadR1 is genomically positioned near genes that are involved in stress response and virulence. In addition, it was previously reported that cdPadR1 and a homologue from the historical C. difficile strain 630 (CD630_1154) were differentially expressed when exposed to stressors, including antibiotics. RESULTS: The crystal structure of cdPadR1 was determined to 1.9 Å resolution, which revealed that it belongs to the PadR-s2 subfamily of PadR transcriptional regulators. cdPadR1 binds its own promoter and other promoter regions from within the C. difficile R20291 genome. DNA binding experiments demonstrated that cdPadR1 binds a region comprised of inverted repeats and an AT-rich core with the predicted specific binding motif, GTACTAT(N2)ATTATA(N)AGTA, within its own promoter that is also present in 200 other regions in the C. difficile R20291 genome. Mutation of the highly conserved W in α4 of the effector binding/oligomerization domain, which is predicted to be involved in multi-drug recognition and dimerization in other PadR-s2 proteins, resulted in alterations of cdPadR1 binding to the predicted binding motif, potentially due to loss of higher order oligomerization. CONCLUSIONS: Our results indicate that cdPadR1 binds a region within its own promoter consisting of the binding motif GTACTAT(N2)ATTATA(N)AGTA and seems to associate non-specifically with longer DNA fragments in vitro, which may facilitate promoter and motif searching. This suggests that cdPadR1 acts as a transcriptional auto-regulator, binding specific sites within its own promoter, and is part of a broad gene regulatory network involved, in part, with environmental stress response, antibiotic resistance and virulence.

Nitric oxide coupling to generate N2O 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 N2O. In order to probe the capacity of a single-heme system to mediate this NO-to-N2O transformation, various scenarios for addition of electrons, protons, and a second NO molecule to a heme nitrosyl to generate N2O 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-N2O product. We also report a hitherto unreported directional second protonation that results in either productive N2O formation with loss of water, or formation of a non-productive hyponitrous acid adduct Fe{HONNOH}.

A bridged di-iron porphyrin hyponitrite complex as a model for biological N2O production from hyponitrite.

Heme-hyponitrites are intermediates that form at the bimetallic active sites of bacterial nitric oxide reductases. To probe a possible effect of the Fe-Fe distance on hyponitrite stability, we prepared a bridged bis-porphyrin Fe-hyponitrite compound, namely [(OEP-CH2)Fe]2(µ2,η(1),η(1)-ONNO). Its υNO of 992 cm(-1) (υ15NO of 976 cm(-1)) is close to the υNO of 983 cm(-1) reported previously by us for the crystallographically characterized [(OEP)Fe]2(µ2,η(1),η(1)-ONNO) compound. The bridged bis-porphyrin Fe-hyponitrite complex is unstable with respect to N2O production, supporting the role of the bis-Fe porphyrin system in hyponitrite conversion to N2O. The preparation and crystallographic determination of the bridging sulfato derivative is also reported.

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 (RNO2; R = alkyl, aryl) to the nitroso RN=O, hydroxylamino RNHOH, or amine RNH2 derivatives. Metronidazole (Mtz) is a nitro-containing antibiotic that is commonly prescribed for lower-gut infections caused by the anaerobic bacterium Clostridium difficile. C. difficile infections rank number one among hospital acquired infections, and can result in diarrhea, severe colitis, or even death. Although NRs have been implicated in Mtz resistance of C. difficile, no NRs have been characterized from the hypervirulent R20291 strain of C. difficile. We report the first expression, purification, and three-dimensional X-ray crystal structures of two NRs from the C. difficile R20291 strain. The X-ray crystal structures of the two NRs were solved to 2.1 Å resolution. Their homodimeric structures exhibit the classic NR α+ß fold, with each protomer binding one FMN cofactor near the dimer interface. Functional assays demonstrate that these two NRs metabolize Mtz with associated re-oxidation of the proteins. Importantly, these results represent the first isolation and characterization of NRs from the hypervirulent R20291 strain of relevance to organic RNO2 (e.g., Mtz) metabolism.

Crystallographic characterization of the nitric oxide derivative of R-state human hemoglobin.

Nitric oxide (NO) is a signaling agent that is biosynthesized in vivo. NO binds to the heme center in human hemoglobin (Hb) to form the HbNO adduct. This reaction of NO with Hb has been studied for many decades. Of continued interest has been the effect that the bound NO ligand has on the geometrical parameters of the resulting heme-NO active site. Although the crystal structure of a T-state human HbNO complex has been published previously, that of the high affinity R-state HbNO derivative has not been reported to date. We have crystallized and solved the three-dimensional X-ray structure of R-state human HbNO to 1.90 Å resolution. The differences in the FeNO bond parameters and H-bonding patterns between the α and ß subunits contribute to understanding of the observed enhanced stability of the α(FeNO) moieties relative to the ß(FeNO) moieties in human R-state HbNO.

Synthesis, molecular structure, and spectroelectrochemistry of a nitrosyl iron porphyrin containing an unsymmetrical xanthene-linked porphyrin core.

Synthetic nitrosyl porphyrins with meso-aryl substituents are potential models for the biologically-important NO-bound P460 heme cofactor. A five-coordinate iron nitrosyl tetraaryl-porphyrin (HTPPX-CO2H)Fe(NO) containing a xanthene-based meso substituent has been prepared. The crystal structure of this formally {FeNO}7 complex reveals an ordered axial and bent NO ligand (∠FeNO=142.5(6)Å) displaying an off-axis tilt of the nitrosyl N atom from the heme normal by 9.2°. Surprisingly, the porphyrin core does not display the expected asymmetry in FeN(por) distances frequently observed in iron nitrosyl porphyrins. The redox behavior as determined by cyclic voltammetry reveals, in contrast to most (por)Fe(NO) compounds, a fast NO dissociation after electrooxidation in CH2Cl2 to result in a net chemically-irreversible oxidation at Epa=+0.77V vs Ag/AgCl. IR spectroelectrochemistry reveals a recombination, on the spectroelectrochemistry time-scale, of the dissociated NO on oxidation with electrogenerated [(HTPPX-CO2H)Fe]+.

Characterization of the bridged hyponitrite complex {[Fe(OEP)](2)(µ-N(2)O(2))}: reactivity of hyponitrite complexes and biological relevance.

The detoxification of nitric oxide (NO) by bacterial NO reductase (NorBC) represents a paradigm of how NO can be detoxified anaerobically in cells. In order to elucidate the mechanism of this enzyme, model complexes provide a convenient means to assess potential reaction intermediates. In particular, there have been many proposed mechanisms that invoke the formation of a hyponitrite bridge between the heme b3 and nonheme iron (FeB) centers within the NorBC active site. However, the reactivity of bridged iron hyponitrite complexes has not been investigated much in the literature. The model complex {[Fe(OEP)]2(µ-N2O2)} offers a unique opportunity to study the electronic structure and reactivity of such a hyponitrite-bridged complex. Here we report the detailed characterization of {[Fe(OEP)]2(µ-N2O2)} using a combination of IR, nuclear resonance vibrational spectroscopy, electron paramagnetic resonance, and magnetic circular dichroism spectroscopy along with SQUID magnetometry. These results show that the ground-state electronic structure of this complex is best described as having two intermediate-spin (S = (3)/2) iron centers that are weakly antiferromagnetically coupled across the N2O2(2-) bridge. The analogous complex {[Fe(PPDME)]2(µ-N2O2)} shows overall similar properties. Finally, we report the unexpected reaction of {[Fe(OEP)]2(µ-N2O2)} in the presence and absence of 1-methylimidizole to yield [Fe(OEP)(NO)]. Density functional theory calculations are used to rationalize why {[Fe(OEP)]2(µ-N2O2)} cannot be formed directly by dimerization of [Fe(OEP)(NO)] and why only the reverse reaction is observed experimentally. These results thus provide insight into the general reactivity of hyponitrite-bridged iron complexes with general relevance for the N-N bond-forming step in NorBC.

(1-Methyl-1H-imidazole-κN (3))(1-methyl-2-nitroso-benzene-κN)(5,10,15,20-tetra-phenyl-porphyrinato-κ(4) N)iron(II) di-chloro-methane monosolvate.

The solvated title compound, [Fe(C44H28N4)(C4H6N2)(C7H7NO)]·CH2Cl2, is a porphyrin complex containing an octahedrally coordinated Fe(II) atom with 1-methylimidazole [Fe-N = 2.0651 (17) Å] and o-nitro-sotoluene ligands at the axial positions. The o-nitro-sotoluene ligand is N-bound to iron(II) [Fe-N = 1.8406 (18)Å and Fe-N-O = 122.54 (14)°]. The axial N-Fe-N linkage is almost linear, with a bond angle of 177.15 (7)°. One phenyl group of the porphyrin ligand is disordered over two orientations in a 0.710 (3):0.290 (3) ratio. The di-chloro-methane solvent mol-ecule was severely disordered and its contribution to the scattering was removed with the SQUEEZE routine [van der Sluis & Spek (1990 ▶). Acta Cryst. A46, 194-201].

(2,3,5,6-Tetra-fluoro-phenolato)[5,10,15,20-tetra-kis-(4-meth-oxy-phen-yl)porphyrinato]iron(III) cyclo-hexane monosolvate.

The title compound, [Fe(C6HF4O)(C48H36N4O4)]·C6H12, represents a five-coordinate iron(III) porphyrin complex in a square-pyramidal geometry with a tetra-fluoro-phenolate anion as the axial ligand. The Fe(III) atom is displaced by 0.364 (2) Šfrom the 24-atom mean plane of the porphyrinate ring towards the tetra-fluoro phenolate anion. The average Fe-N distance is 2.053 (2) Šand the Fe-O distance is 1.883 (2) Å. A porphyrin aryl H atom points in the general direction of the phenoxide ring. The mean plane separation between the 24-atom porphyrin planes of two adjacent porphyrin rings is ∼3.7 Å, and the lateral shift is ∼3.5 Šresu, ting in an Fe⋯Fe separation of 5.6167 (14) Å.

(2,3,5,6-Tetra-fluoro-phenolato-κO)(5,10,15,20-tetra-phenyl-porphyrinato)iron(III).

The title compound, [Fe(C44H28N4)(C6HF4O)], is a porphyrin complex with iron(III) in fivefold coordination with a tetra-fluoro-phenolate group as the axial ligand. The Fe atom and the phenolate ligand are disordered across the porphyrin ring with the two phenolates appearing to be roughly related by a center of symmetry. The occupancies of the two phenolate groups refined to 0.788 (3) for the major component and 0.212 (3) for the minor component. The structure shows extraordinary Fe displacements of 0.488 (4) (major) and 0.673 (4) Š(minor) from the 24-atom mean plane of the porphyrin. The Fe-Np distances range from 2.063 (4) to 2.187 (6) Šand the Fe-O distances are 1.903 (5) Šfor major component and 1.87 (2) Šfor minor component. The four phenyl groups attached to the porphyrin ring form dihedral angles of 63.4 (4), 49.6 (4), 62.4 (4), and 63.3 (4)° (in increasing numerical order) with the three nearest C atoms of the porphyrin ring. The major and minor component phenolate groups form dihedral angles of 24.9 (4)° and 24.8 (4)°, respectively, with the four porphyrin N atoms. The Fe⋯Fe distance between the two iron(III) atoms of adjacent porphyrin mol-ecules is 6.677 (3) Å. No close inter-molecular inter-action was observed. The crystal studied was twinned by inversion, with a major-minor component ratio of 0.53 (3):0.47 (3).