H-bonding Residues Research Articles

Pore Restructuring of Peptide Frameworks by Mutations at Distal Packing Residues.

Porous framework materials are highly useful for catalysis, adsorption, and separations. Though they are usually made from inorganic and organic building blocks, recently, folded peptides have been utilized for constructing frameworks, opening up an enormous structure-space for exploration. These peptides assemble in a metal-free fashion using π-stacking, H-bonding, dispersion forces, and the hydrophobic effect. Manipulation of pore-defining H-bonding residues is known to generate new topologies, but the impact of mutations in the hydrophobic packing region facing away from the pores is less obvious. To explore their effects, we synthesized variants of peptide frameworks with mutations in the hydrophobic packing positions and found by single-crystal X-ray crystallography (SC-XRD) that they induce significant changes to the framework pore structure. These structural changes are driven by a need to maximize van der Waals interactions of the nonpolar groups, which are achieved by various mechanisms including helix twisting, chain flipping, chain offsetting, and desymmetrization. Even subtle changes to the van der Waals interface, such as the introduction of a methyl group or isomeric replacement, result in significant pore restructuring. This study shows that the dispersion interactions upholding a peptide material are a rich area for structural engineering.

Cofactor Dynamics Couples the Protein Surface to the Heme in Cytochrome c, Facilitating Electron Transfer.

Electron transport through biomolecules and in biological transport networks is of great importance to bioenergetics and biocatalysis. More generally, it is of crucial importance to understand how the pathways that connect buried metallocofactors to other cofactors, and to protein surfaces, affect the biological chemistry of metalloproteins. In terms of electron transfer (ET), the strongest coupling pathways usually comprise covalent and hydrogen bonded networks, with a limited number of through-space contacts. Herein, we set out to determine the relative roles of hydrogen bonds involved in ET via an established heme-to-surface tunneling pathway in cytochrome (cyt) c (i.e., heme-W59-D60-E61-N62). A series of cyt c variants were produced where a ruthenium tris(diimine) photooxidant was placed at position 62 via covalent modification of the N62C residue. Surprisingly, variants where the H-bonding residues W59 and D60 were replaced (i.e., W59F and D60A) showed no change in ET rate from the ferrous heme to Ru(III). In contrast, changing the composition of an alternative tunneling pathway (i.e., heme-M64-N63-C62) with the M64L substitution shows a factor of 2 decrease in the rate of heme-to-Ru ET. This pathway involves a through-space tunneling step between the heme and M64 residue, and such steps are usually disfavored. To rationalize why the heme-M64-N63-C62 is preferred, molecular dynamics (MD) simulations and Pathways analysis were employed. These simulations show that the change in heme-Ru ET rates is attributed to different conformations with compressed donor-acceptor distances, by ∼2 Å in pathway distance, in the M64-containing protein as compared to the M64L protein. The change in distance is correlated with changes in the electronic coupling that are in accord with the experimentally observed heme-Ru ET rates. Remarkably, the M64L variation at the core of the protein translates to changes in cofactor dynamics at the protein surface. The surface changes identified by MD simulations include dynamic anion-π and dipole-dipole interactions. These interactions influence the strength of tunneling pathways and ET rates by facilitating decreases in through-space tunneling distances in key coupling pathways.

Histidine-Gated Proton-Coupled Electron Transfer to the CuA Site of Nitrous Oxide Reductase.

Copper-containing nitrous oxide reductase (N2OR) is the only known enzyme to catalyze the conversion of the environmentally critical greenhouse gas nitrous oxide (N2O) to dinitrogen (N2) as the final step of bacterial denitrification. Other than its unique tetranuclear active site CuZ, the binuclear electron entry point CuA is also utilized in other enzymes, including cytochrome c oxidase. In the CuA site of Pseudomonas stutzeri N2OR, a histidine ligand was found to undergo a conformational flip upon binding of the substrate N2O between the two copper centers. Here we report on the systematic mutagenesis and spectroscopic and structural characterization of this histidine and surrounding H-bonding residues, based on an established functional expression system for PsN2OR in E. coli. A single hydrogen bond from Ser550 is sufficient to stabilize an unbound conformation of His583, as shown in a Asp576Ala variant, while the additional removal of the hydrogen bond in a Asp576Ala/Ser550Ala double variant compelled His583 to stay in a bound conformation as a ligand to CuA. Systematic mutagenesis of His583 to Ala, Asp, Asn, Glu, Gln, Lys, Phe, Tyr, and Trp showed that although both the CuZ and CuA sites were present in all the variants, only the ones with a protonable side chain, i.e., His, Asp, and Glu, were able to mediate electron transfer at physiological pH. This observation is in line with a proton-coupled electron transfer mechanism at the CuA site of N2OR.

Structural and functional characterization of the pore-forming domain of pinholin S2168

Pinholin S2168 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual "pinholes." Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state 15N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (Smol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G40xxxS44xxxG48) together with a patch of H-bonding residues (T51, T54, N55) sideways along TMD2, available for helix-helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Efficient green approaches for the preparation of physically crosslinked chitin gel materials by freeze-induced self-assembly

Chitin derived from crustacean biomass has great potential for the production of eco-friendly materials. However, one challenge is its insolubility in regular solvents. Herein, the gelation of chitin alkali suspension was induced via freeze-thawing approach. The as-prepared hydrogels with/without dialysis treatment were freeze-dried into solid foams to display “dry durian fruit” appearance. It was found that dialysis did influence the properties, in which the dialyzed samples exhibited superior water content, swelling ratio, porosity, thermal stability, and smoother surface morphological results. Moreover, the dialyzed chitin foams exhibited nearly the same level of thermal stability compared to chitin powder. With respect to formation mechanisms, we propose that alkali treatment swells chitin and the freezing process deconstructed and weakened H-bonding, while the weakened H-bonding residue self-assembly contributed to hydrogel formation. This research represents a simple and green route for converting chitin suspension to gel materials.

Role of 2nd sphere H-bonding residues in tuning the kinetics of CO2 reduction to CO by iron porphyrin complexes.

Iron porphyrins are potential catalysts for the electrocatalytic and photocatalytic reduction of CO2. It has been recently established that the reduction of CO2 by an iron porphyrin complex with a hydrogen bonding distal pocket involves at least two intermediates: a Fe(ii)-CO22- and a Fe(ii)-COOH species. A distal hydrogen bonding interaction was found to be key in determining the stability of these intermediates and affecting both the selectivity and rate of CO2 reduction. In this report, a series of iron porphyrins that vary only in the distal H-bonding network are further investigated and these exhibit turnover frequencies (TOFs) ranging from 1.0 s-1 to 103 s-1. The experimental TOFs correlate with the H-bonding ability of the distal superstructure of these iron porphyrin complexes and analysis suggests that H-bonding alone can tune the rate of CO2 reduction by as much as 1000 fold. DFT calculations provide a detailed insight into how the, apparently weak, 2nd sphere interactions lead to efficient CO2 activation for reduction. The ability to tune CO2 reduction rates by changing the H-bonding residue instead of the acid source is a convenient way to tune CO2 reduction electrocatalysis without compromising selectivity by introducing competitive hydrogen evolution reaction or formate generation.

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Proton pumping A-type cytochrome c oxidase (CcO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type CcOs, studies of bovine mitochondrial CcO have led to suggestions that its H channel instead provides this route. Here, we have studied H-channel function by performing atomistic molecular dynamics simulations on the entire, as well as core, structure of bovine CcO in a lipid-solvent environment. The majority of residues in the H channel do not undergo large conformational fluctuations. Its upper and middle regions have adequate hydration and H-bonding residues to form potential proton-conducting channels, and Asp51 exhibits conformational fluctuations that have been observed crystallographically. In contrast, throughout the simulations, we do not observe transient water networks that could support proton transfer from the N phase toward heme a via neutral His413, regardless of a labile H bond between Ser382 and the hydroxyethylfarnesyl group of heme a In fact, the region around His413 only became sufficiently hydrated when His413 was fixed in its protonated imidazolium state, but its calculated pKa is too low for this to provide the means to create a proton transfer pathway. Our simulations show that the electric dipole moment of residues around heme a changes with the redox state, hence suggesting that the H channel could play a more general role as a dielectric well.

A competing hydrophobic tug on L596 to the membrane core unlatches S4–S5 linker elbow from TRP helix and allows TRPV4 channel to open

We have some generalized physical understanding of how ion channels interact with surrounding lipids but few detailed descriptions on how interactions of particular amino acids with contacting lipids may regulate gating. Here we discovered a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (transient receptor potential vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between the indole of W733 of the TRP helix and the backbone oxygen of L596 secures the helix/linker contact, which acts as a latch maintaining channel closure. The modeled side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model-an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Making this residue less hydrophobic (L596A/G/W/Q/K) reduces open probability [Po; loss-of-function (LOF)], likely due to altered interactions at the polar-nonpolar interface. L596I raises Po [gain-of-function (GOF)], apparently by placing its methyl group further inward and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the levels of α-carbons of H-bonded residues L596 and W733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP helix latch, respectively. These results highlight that L596 lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.

MHCII with full peptide occupancy is a substrate for HLA-DM catalyzed peptide exchange

Abstract The major histocompatibility complex class II (MHCII) antigen-processing pathway is critical for adaptive immunity as the mechanism through which ligands are generated for recognition by CD4 T cells. HLA-DM edits the repertoire of peptides that are presented to T cells by catalyzing multiple rounds of peptide exchange on MHCII molecules. The catalytic potency of DM varies such that peptide exchange is markedly enhanced on some peptide-MHC (pMHC) complexes and not others. This capacity to differentially edit complexes influences specificity and immunodominance in T cell responses. The catalytic mechanism and rules that govern DM susceptibility are not fully understood. Evidence suggests that partial occupancy at the N-terminus of bound peptides is critical for complex sensitivity to DM. We employed a fluorescence polarization assay to independently assess the effective affinity and catalytic turnover of DM, components of the catalytic mechanism, for pMHC complexes in real-time kinetic measurements of DM activity. We found that a complex with an N-terminal truncated form of the immunodominate HA peptide, resulting in the loss of three H-bonds mediated by the P-2 and P-1 residues, showed an expected decrease in complex stability and increase in affinity for DM binding, but an unexpected increase in the catalytic turnover when compared to the unmodified HA-MHC complex. This result was even more pronounced when a conserved bidentate H-bonding residue near the P1 anchor was disrupted by site-directed mutagenesis on an HA-MHC complex. Together, this implies that pMHC interactions near the N-terminal segment of a peptide may be intact in the functionally active DM-pMHC catalytic complex and provide insights into the DM catalytic mechanism.

A Lipid-Exposed Residue at the Start of S4-S5 Linker Controls TRPV4 Gating

We have some generalized physical understanding of how ion channels interact with surrounding lipids but lack detailed descriptions on how interactions of particular amino acid with contacting lipids may regulate gating. Here we show a structure-specific interaction between an amino acid and inner-leaflet lipid that governs the gating transformations of TRPV4 (Transient Receptor Potential Vanilloid type 4). Many cation channels use a S4-S5 linker to transmit stimuli to the gate. At the start of TRPV4's linker helix is leucine 596. A hydrogen bond between L596's backbone oxygen and the indole of W733 of the TRP helix acts as a latch to maintain channel closure. The side chain of L596 interacts with the inner lipid leaflet near the polar-nonpolar interface in our model - an interaction that we explored by mutagenesis. We examined the outward currents of TRPV4-expressing Xenopus oocyte upon depolarizations as well as phenotypes of expressing yeast cells. Maintaining its hydrophobicity, L596F and L596V channels appear normal in gating. However, making this residue less hydrophobic (L596A, G, W, Q, K) reduces open probability (Po) (loss-of-function, LOF), likely due to altered interactions at the polar-nonpolar interface. L596I raises Po (gain-of-function, GOF), apparently by placing its methyl group closer to the polar region and receiving stronger water repulsion. Molecular dynamics simulations showed that the distance between the β carbons of H-bonded residues 596 and 733 is shortened in the LOFs and lengthened in the GOFs, strengthening or weakening the linker/TRP-helix latch respectively. These results highlight that, in each lipid-exposed focus, the L596-lipid attraction counteracts the latch bond in a tug-of-war to tune the Po of TRPV4.

Structural Variabilities in β-Lactamase (blaA) of Different Biovars of Yersinia enterocolitica: Implications for β-Lactam Antibiotic and β-Lactamase Inhibitor Susceptibilities.

Yersiniosis caused by Yersinia enterocolitica has been reported from all continents. The bacterial species is divided into more than fifty serovars and six biovars viz. 1A, 1B, 2, 3, 4 and 5 which differ in geographical distribution, ecological niches and pathogenicity. Most Y.enterocolitica strains harbor chromosomal genes for two β-lactamases, blaA an Ambler class A penicillinase and blaB an Ambler class C inducible cephalosporinase. In the present study, susceptibility to b-lactam antibiotics and β-lactamase inhibitor was studied for Y. enterocolitica strains of biovars 1A, 1B, 2 and 4. We observed that β-lactamases were expressed differentially among strains of different biovars. To understand the molecular mechanisms underlying such differential expression, the sequences of genes and promoters of blaA were compared. Also, the variants of blaA present in different biovars were modeled and docked with amoxicillin and clavulanic acid. The mRNA secondary structures of blaA variants were also predicted in-silico. Our findings indicated that neither variations in the promoter regions, nor the secondary structures of mRNA contributed to higher/lower expression of blaA in different biovars. Analysis of H-bonding residues of blaA variants with amoxicillin and clavulanic acid revealed that if amino acid residues of a β-lactamase interacting with amoxicillin and the clavulanic acid were similar, clavulanic acid was effective in engaging the enzyme, accounting for a significant reduction in MIC of amoxicillin-clavulanate. This finding might aid in designing better β-lactamase inhibitors with improved efficiencies in future.

Molecular Dynamics Simulations of Calcium Pump Structural Disorder

To characterize the conformational dynamics of sarcoplasmic reticulum (SR) calcium pump (SERCA) we performed molecular dynamics simulations beginning with several different high-resolution structures. We quantified differences in structural disorder and dynamics for an open E1 conformation of SERCA (pdb: 1SU4) versus closed E2 structures (pdb: 2ZBD, 1WPG), and observed that dynamic motions of SERCA cytoplasmic domains increased with domain-domain separation distance. The results are useful for interpretation of recent intramolecular Forster resonance energy transfer (FRET) distance constraints obtained for SERCA fused to fluorescent protein tags. Those previous physical measurements quantified the population of discrete structural substates and revealed that open conformations of SERCA are more dynamically disordered than compact conformations. The present simulations support this hypothesis and provide additional details of SERCA structural dynamics. Specifically, all-atoms simulations revealed large scale translational and rotational motions of the SERCA N-, A- and P-domains. We also observed a transition from an open to a closed headpiece conformation over the course of a 400 ns trajectory. Intermediate structures sampled during the open-to-closed transition were long lived, with >50 ns dwell times observed for each conformation. We observed well-resolved peaks in a histogram of calculated domain separation distances, consistent with saltatory transitions between discrete conformational substates. The open-to-closed conformational transition was mediated in part by an initial interaction of an N-domain loop (Nbeta5-beta6, residues 426-436) that formed dynamic H-bonds with residues 133-139 of the A-domain. The propensity for domain closure was increased by pre-alignment of H-bonding residues and decreased by mutation of these residues to Ala. Overall the data suggest that N-domain loop residues are particularly important for mediating the transition through several novel structural substates during the closure of the SERCA cytoplasmic headpiece.

13C structuring shifts for the analysis of model β-hairpins and β-sheets in proteins: diagnostic shifts appear only at the cross-strand H-bonded residues

The present studies have shown that (13)C=O, (13)C(α) and (13)C(β) of H-bonded strand residues in β-hairpins provide additional probes for quantitating the extent of folding in β-hairpins and other β-sheet models. Large differences in the structuring shifts (CSDs) of these (13)C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three (13)C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from (13)C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the (13)C' and (13)C(β) CSDs will provide the best differentiation with 100%-folded CSD values approaching -2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein β-sheets. Our survey of reported (13)C shifts in β-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.

Insertion of an H‐Bonding Residue into the Distal Pocket of the Ferriheme Protein Nitrophorin 4: Effect on NitriteIron Coordination and Nitrite Disproportionation

Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein-nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G-H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H(2)O molecules from the heme cavity prevents the proton donation pathway through Asp30.

Nitrite reduction by Co II and Mn II substituted myoglobins : Towards understanding necessary components of Mb nitrite reductase activity

Nitrite reduction to nitric oxide by heme proteins is drawing increasing attention as a protective mechanism to hypoxic injury in mammalian physiology. Here we probe the nitrite reductase (NiR) activities of manganese(II)- and cobalt(II)-substituted myoglobins, and compare with data obtained previously for the iron(II) analog wt Mb II. Both Mn IIMb and Co IIMb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H +], as previously determined for the Fe II analog wt Mb II. The second order rate constants ( k 2 ) at pH 7.4 and T = 25 °C, were 0.0066 and 0.015 M − 1 s − 1 for Co IIMb and Mn IIMb, respectively, both orders of magnitude slower than the k 2 (6 M − 1 s − 1 ) for wt Mb II. The final reaction products for Mn IIMb consisted of a mixture of the nitrosyl Mn IIMb(NO) and Mn IIIMb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co IIMb were found to be the nitrito complex Co IIIMb(ONO −) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co IIIMb as reflected in the respective M IIIMb(ONO −) formation constants K nitrite : 2100 M − 1 (Co III) and <~0.4 M − 1 (Mn III). We also report the formation constants (3.7 and 30 M − 1 , respectively) for the nitrite complexes of the mutant metmyoglobins H64V Mb III(NO 2 −) and H64V/V67R Mb III(ONO −) and a K nitrite revised value (120 M − 1 ) for the nitrite complex of wt metMb. The respective K nitrite values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the Mb III distal pocket or the Arg67 in H64V/V67R Mb III, in stabilizing nitrite coordination. Notably, the NiR activities of the corresponding ferrous Mbs follow a similar sequence suggesting that nitrite binding to these centers are analogously affected by the H-bonding residues.

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600–620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450–460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin ( Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.

Linkage Isomerization in Heme−NOx Compounds: Understanding NO, Nitrite, and Hyponitrite Interactions with Iron Porphyrins

Nitric oxide (NO) and its derivatives such as nitrite and hyponitrite are biologically important species of relevance to human health. Much of their physiological relevance stems from their interactions with the iron centers in heme proteins. The chemical reactivities displayed by the heme-NOx species (NOx = NO, nitrite, hyponitrite) are a function of the binding modes of the NOx ligands. Hence, an understanding of the types of binding modes extant in heme-NOx compounds is important if we are to unravel the inherent chemical properties of these NOx metabolites. In this Forum Article, the experimentally characterized linkage isomers of heme-NOx models and proteins are presented and reviewed. Nitrosyl linkage isomers of synthetic iron and ruthenium porphyrins have been generated by photolysis at low temperatures and characterized by spectroscopy and density functional theory calculations. Nitrite linkage isomers in synthetic metalloporphyrin derivatives have been generated from photolysis experiments and in low-temperature matrices. In the case of nitrite adducts of heme proteins, both N and O binding have been determined crystallographically, and the role of the distal H-bonding residue in myoglobin in directing the O-binding mode of nitrite has been explored using mutagenesis. To date, only one synthetic metalloporphyrin complex containing a hyponitrite ligand (displaying an O-binding mode) has been characterized by crystallography. This is contrasted with other hyponitrite binding modes experimentally determined for coordination compounds and computationally for NO reductase enzymes. Although linkage isomerism in heme-NOx derivatives is still in its infancy, opportunities now exist for a detailed exploration of the existence and stabilities of the metastable states in both heme models and heme proteins.

The Distal Pocket Histidine Residue in Horse Heart Myoglobin Directs the O-Binding Mode of Nitrite to the Heme Iron

It is now well-established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example, the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO(2)(-)) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMb(III) (i.e., ferric Mb(III)) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMb(III) H64V (2.0 A resolution) and its nitrite ion adduct metMb(III) H64V-nitrite (1.95 A resolution), and metMb(III) H64V/V67R (1.9 A resolution) and its nitrite ion adduct metMb(III) H64V/V67R-nitrite (2.0 A resolution). These are compared to the known structures of wild-type (wt) hh metMb(III) and its nitrite ion adduct hh metMb(III)-nitrite, which binds NO(2)(-) via an O-atom in a trans-FeONO configuration. Unlike wt metMb(III), no axial H(2)O is evident in either of the metMb(III) mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMb(III)s with cysteine as a reducing agent and of the (ferrous) Mb(II)s with nitrite ion under anaerobic conditions. The Mb(II)s were generated by reduction of the Mb(III) precursors in a second-order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the Mb(II)s by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

DFT calculations of comparative energetics and ENDOR/Mössbauer properties for two protonation states of the iron dimer cluster of ribonucleotide reductase intermediate X.

Two models (I and II) for the active site structure of class-I ribonucleotide reductase (RNR) intermediate X in subunit R2 have been studied in this paper, using broken-symmetry density functional theory (DFT) incorporated with the conductor like screening (COSMO) solvation model and with the finite-difference Poisson-Boltzmann self-consistent reaction field (PB-SCRF) calculations. Only one of the bridging groups between the two iron centers is different between model-I and model-II. Model-I contains two mu-oxo bridges, while model-II has one bridging oxo and one bridging hydroxo. These are large active site models including up to the fourth coordination shell H-bonding residues. Mössbauer and ENDOR hyperfine property calculations show that model-I is more likely to represent the active site structure of RNR-X. However, energetically our pK(a) calculations at first highly favored the bridging oxo and hydroxo (in model-II) structure of the diiron center rather than having the di-oxo bridge (in model-I). Since the Arg236 and the nearby Lys42, which are very close to the diiron center, are on the protein surface of RNR-R2, it is highly feasible that one or two anion groups in solution would interact with the positively charged side chains of Arg236 and Lys42. The anion group(s) can be a reductant, phosphate, sulfate, nitrate, and other negatively charged groups existing in biological environments or in the buffer of the experiment. Since sulfate ions certainly exist in the buffer of the ENDOR experiment, we have examined the effect of the sulfate (SO(4)(2-), surrounded by explicit water molecules) H-bonding to the side chain of Arg236. We find that when sulfate interacts with Arg236, the carboxylate group of Asp237 tends to be protonated, and once Asp237 is protonated, the Fe(iii)Fe(iv) center in X favors the di-oxo bridge (model-I). This would explain that the ENDOR observed RNR-X active site structure is likely to be represented by model-I rather than model-II.

Ambidentate H-bonding by heme-bound NO: structural and spectral effects of –O versus –N H-bonding

Resonance Raman studies have uncovered puzzling complexities in the structures of NO adducts of heme proteins. Although CO adducts of heme proteins obey well-behaved anti-correlations between Fe-C and C-O stretching frequencies, which reflect changes in backbonding induced by distal H-bonding residues, the corresponding NO data are scattered. This scatter can be traced to distal influences, since protein-free NO-hemes do show well-behaved anti-correlations. Why do distal effects produce irregularities in vFeN/ vNO plots but not in vFeC/vCO plots? We show via density functional theory (DFT) computations on model systems that the response to distal H-bonding differs markedly when the NO acceptor atom is N versus O. Backbonding is augmented by H-bonding to O, but the effect of H-bonding to N is to weaken both N-O and N-Fe bonds. The resulting downward deviation from the vFeN/vNO backbonding line increases with increasing H-bond strength. This effect explains the deviations observed for a series of myoglobin variants, in which the strength of distal H-bonding is modulated by distal pocket residue substitutions. Most of the data follow a positive vFeN/vNO correlation with the same slope as that calculated for H-bonding to N. Such deviations are not observed for CO adducts, because the CO pi* orbital is unoccupied, and serves as a delocalized acceptor of H-bonds. H-bonding to N primes NO-heme for reduction to the HNO adduct, a putative intermediate in NO-reducing enzymes.