Cα Cβ Bond Research Articles

New preorganized γ-amino acids as foldamer building blocks.

An asymmetric synthesis of two new diastereomeric γ-amino acids is described. Both molecules contain a cyclohexyl ring to limit conformational flexibility about the Cα-Cβ bond; they differ in having cis vs trans stereochemistry on the ring. Residues derived from the cis γ isomer are shown to support helical secondary structures in α/γ-peptide oligomers.

Oxygen radical-mediated oxidation reactions of an alanine peptide motif - density functional theory and transition state theory study

BackgroundOxygen-base (O-base) oxidation in protein backbone is important in the protein backbone fragmentation due to the attack from reactive oxygen species (ROS). In this study, an alanine peptide was used model system to investigate this O-base oxidation by employing density functional theory (DFT) calculations combining with continuum solvent model. Detailed reaction steps were analyzed along with their reaction rate constants.ResultsMost of the O-base oxidation reactions for this alanine peptide are exothermic except for the bond-breakage of the Cα-N bond to form hydroperoxy alanine radical. Among the reactions investigated in this study, the activated energy of OH α-H abstraction is the lowest one, while the generation of alkylperoxy peptide radical must overcome the highest energy barrier. The aqueous situation facilitates the oxidation reactions to generate hydroxyl alanine peptide derivatives except for the fragmentations of alkoxyl alanine peptide radical. The Cα-Cβ bond of the alkoxyl alanine peptide radical is more labile than the peptide bond.Conclusionthe rate-determining step of oxidation in protein backbone is the generation of hydroperoxy peptide radical via the reaction of alkylperoxy peptide radical with HO2. The stabilities of alkylperoxy peptide radical and complex of alkylperoxy peptide radical with HO2 are crucial in this O-base oxidation reaction.

Intramolecular hydrogen atom migration along the backbone of cationic and neutral radical tripeptides and subsequent radical-induced dissociations

Dissociation of peptide radical ions involves competition between charge-induced and radical-induced reactions that can be preceded by isomerization. The isomeric radical cations of the peptide methyl ester [G˙GR-OMe](+) and [GG˙R-OMe](+) provide very similar collision-induced dissociation (CID) spectra, suggesting that isomerization occurs prior to fragmentation. They undergo characteristic radical-induced bond cleavage of the peptide N-terminal amide bond resulting in the y2(+) ion, and of the arginine side-chain's Cα-Cβ bond giving protonated allylguanidine {[CH2[double bond, length as m-dash]CHCH2NHC(NH2)2](+), m/z 100}. The absence of a y2(+) fragment ion in the CID of the radical cationic tripeptide [ACH3G˙R](+) and of an m/z 100 ion in the spectrum of [G˙ACH3R](+) (where ACH3 is an α-aminoisobutyric acid residue, which cannot form an α-carbon-centered radical through hydrogen atom transfer) establishes the importance of hydrogen atom migration along the peptide backbone prior to specific radical-induced fragmentations. Herein we use density functional theory (DFT) at the B3LYP/6-31++G(d,p) level to evaluate the barriers for interconversion between the α-carbon-centered radicals and for dissociation. The radical cations [G˙GR](+) and [GG˙R](+) have their radicals located on the α-carbon atoms of the peptide backbone and their charge densities largely sequestered on the guanidine groups of the side-chain of arginine residues. This is in contrast to the isomeric radical cations of [GGG]˙(+), in which the charge resides necessarily on the peptide backbone. The lower charge densities on the backbones of [G˙GR](+) and [GG˙R](+) result in greater structural flexibility, decreasing the barrier for interconversion between these α-carbon-centered radicals to 36.2 kcal mol(-1) (cf. 44.7 kcal mol(-1) for [GGG]˙(+)). The total absence of charge, assessed by examining intramolecular hydrogen atom transfers among the three α-carbon centers of the isomeric neutral α-carbon-centered triglycine radicals [GGG-H]˙, leads to an additional but slight reduction in enthalpy, to approximately 34 kcal mol(-1).

Mechanism for folate‐independent aldolase reaction catalyzed by serine hydroxymethyltransferase

Serine hydroxymethyltransferase catalyzes the cleavage of β-hydroxyamino acids into glycine and aldehydes in the absence of tetrahydrofolate. The enzyme accepts various β-hydroxyamino acids as the substrate of this reaction. The reaction rate varies depending on the substituent and stereochemistry at the Cβ atom: the erythro forms and the β-phenyl substituent are preferred over the threo forms and the β-methyl substituent, respectively. Although several mechanisms have been proposed, what determines the substrate preference remains unclear. We first performed quantum mechanical calculations to assess the validity of the reaction mechanisms. The results indicate that the retro-aldol mechanism starting with abstraction of the proton from the β-hydroxyl group is plausible. This also suggests that Cα-Cβ bond cleavage is the rate-limiting step. We next measured the dependence of the rate constants on temperature with four representative substrates and calculated the activation energies and pre-exponential factors from the Arrhenius plots. The activation energies of the erythro forms were lower than those of the threo forms. The β-phenyl substituent lowered the activation energy in the threo form, whereas it did not alter the activation energy but increased the pre-exponential factor in the erythro form. We present a unified model to explain the origin of the substituent and stereochemical preferences by combining the theoretical and experimental results. A possible biological role of the tetrahydrofolate-independent activity in thermophiles is also discussed.

Study on Mechanism of Action of Catalysts on Liquefaction of Bagasse Alkali Lignin

Bagasse alkali lignin is the primary constituent of pulping black liquor. The phenolic products such as phenol, 2,6-dimethoxy-phenol and 2-methoxy-phenol were obtained by catalytic liquefaction from bagasse alkali lignin, with the aim of enhancing its use value. In the investigation, under optimal conditions, using SiO2-Al2O3 or FeS as catalyst, the phenols yield reached 54.10% and 55.18%, respectively. HPLC spectrum showed that the phenolic products of them were different. FT-IR and H-NMR spectrums showed that, using SiO2-Al2O3 as the catalyst, the α-O-4 and β-O-4 ether bonds and C-Cα bonds of lignin units were prone to break, forming 2,6-dimethoxy-phenol, 2-methoxy-phenol and phenol. While FeS action was more difficult, the α-O-4 and β-O-4 ether bonds and Cα-Cβ bonds of lignin units broke down. Then, the introduction of additional hydroxyl groups from glycols consequently increased the final product solubility. At the same time, Cα–C bond of the phenylpropane linkage was broken to produce phenolic chemicals. Third, part of the phenylpropane linkage was prone to break Cα–Cβ bond and structural rearrangement occurred. So the content of 2-methoxy-4-methyl-phenol and the total yield of phenols were high.

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

Unexpected Tricovalent Binding Mode of Boronic Acids within the Active Site of a Penicillin-Binding Protein

Boronic acids bearing appropriate side chains are good inhibitors of serine amidohydrolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the active site and mimicking the transition state of the enzymatic reaction. We have solved the structures of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethylboronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents the first tricovalent enzyme-inhibitor adducts observed by crystallography. In two of the five R39-boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent complex from a classical monocovalent complex may involve rotation around the Ser49 Cα-Cβ bond to place the boron in a position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.

Identification of DypB from Rhodococcus jostii RHA1 as a Lignin Peroxidase

Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn(2+) by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl(2). Incubation of recombinant DypB with a β-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C(α)-C(β) bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn(II) (k(obs) = 2.35 s(-1)) or the β-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn(II) and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.

3-[2-(1H-1,3-Benzodiazol-2-yl)ethyl]-1,3-oxazolidin-2-one

In the title compound, C12H13N3O2, the dihedral angle between the oxazolone ring and the benzimidazole unit is 45.0 (5)°, exhibiting a staggered conformation at the Cα—Cβ bond. In the crystal, a strong N—H⋯N hydrogen bond links the mol­ecules into a C(4) chain along the c axis while a C—H⋯O hydrogen-bonding inter­action generates a C(5) chain along the a axis, i.e. perpendicular to the other chain.

ESR study of structural characteristics and intramolecular motions of tris(3,5-di-tert-butyl-4-hydroxybenzyl)amine free radical in toluene

The magnetic and dynamic properties of tris(2,6-di-tert-butyl-4-hydroxybenzyl)amine free radical was studied by ESR spectroscopy in the temperature range from 170 to 350 K in toluene. Symmetrical conformers in the rigid toluene matrix with characteristic angles θ1 = 26° and θ2 = 60° between the symmetry axis of the 2pz-orbital on the Cα atom of the phenoxyl ring and the direction of the Cα-Cβ bond of the CH2 fragment were determined. The activation energy of transition between the conformers was determined to be 8.2 kcal mol−1. The correlation time of transition between the conformers was established as 8.2·10−1 s.

Carbon-13 CP/MAS solid state NMR and X-ray diffraction spectroscopy studies on lime wood decayed by Chaetomium globosum

The degradation of the plant cell wall by microorganisms has been studied by X-ray diffraction, and solid state CP/MAS 13C NMR spectroscopy. Lime wood ( Tilia cordata Miller) samples were inoculated with Chaetomium globosum for various durations up to 133 days. Structural modifications were assessed by comparing decayed lime wood samples with reference. These methods proved to be able to give insights into the modifications at a molecular level of the cell wall components by Ascomycetes fungi attack. Significant changes in relative crystallinity and apparent lateral crystallite size, as measured by XRD, were detectable relatively early in the decay process. The content of hemicelluloses and cellulose in the wood samples decreased after biodegradation with Chaetomium globosum. The main chemical changes in wood during decay are the loss of hemicelluloses and cellulose simultaneously with lesser changes in lignin structure, which mainly consist in partial loss of methoxyl groups and Cα–Cβ bond cleavage and loss of β-O-4 linkages.

Reactions of an Aromatic σ,σ-Biradical with Amino Acids and Dipeptides in the Gas Phase

Gas-phase reactivity of a positively charged aromatic σ,σ-biradical (N-methyl-6,8-didehydroquinolinium) was examined toward six aliphatic amino acids and 15 dipeptides by using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR) and laser-induced acoustic desorption (LIAD). While previous studies have revealed that H-atom and NH(2) abstractions dominate the reactions of related monoradicals with aliphatic amino acids and small peptides, several additional, unprecedented reaction pathways were observed for the reactions of the biradical. For amino acids, these are 2H-atom abstraction, H(2)O abstraction, addition - CO(2), addition - HCOOH, and formation of a stable adduct. The biradical reacts with aliphatic dipeptides similarly as with aliphatic amino acids, but undergoes also one additional reaction pathway, addition/C-terminal amino acid elimination (addition - CO - NHCHR(C)). These reactions are initiated by H-atom abstraction by the biradical from the amino acid or peptide, or nucleophilic addition of an NH(2) or a HO group of the amino acid or peptide at the radical site at C-6 in the biradical. Reactions of the unquenched C-8 radical site then yield the products not observed for related monoradicals. The biradical reacts with aromatic dipeptides with an aromatic ring in N-terminus (i.e., Tyr-Leu, Phe-Val, and Phe-Pro) similarly as with aliphatic dipeptides. However, for those aromatic dipeptides that contain an aromatic ring in the C-terminus (i.e., Leu-Tyr and Ala-Phe), one additional pathway, addition/N-terminal amino acid elimination (addition - CO - NHCHR(N)), was observed. This reaction is likely initiated by radical addition of the biradical at the aromatic ring in the C-terminus. Related monoradicals add to aromatic amino acids and small peptides, which is followed by Cα-Cβ bond cleavage, resulting in side-chain abstraction by the radical. For biradicals, with one unquenched radical site after the initial addition, the reaction ultimately results in the loss of the N-terminal amino acid. Similar to monoradicals, the C-S bond in amino acids and dipeptides was found to be especially susceptible to biradical attack.

Peripatetic proteins

Peripatetic proteins

Formation of vanillin and syringaldehyde in an oxygen delignification process

The formation of vanillin and syringaldehyde during an oxygen delignification process was evaluated. The cleavage of conjugated Cα-Cβ bonds in phenolic lignin caused by the attack of oxygen leads to the formation of aromatic aldehydes and ketones. The formation mechanism was confirmed by the organic compounds identified in the spent liquor by GC/MS. Additionally, the formation of aromatic aldehydes and ketones of hardwood, softwood, and grass was investigated. The results showed that the formed aromatic aldehydes and ketones were related to the units of lignin structure. Among the aromatic aldehydes and ketones present in the spent liquor from oxygen delignification, vanillin and syringaldehyde are of high content, making the spent liquor a potential resource for vanillin and syringaldehyde production.

Catalytic Activity of the Anaerobic Tyrosine Lyase Required for Thiamine Biosynthesis in Escherichia coli

Thiazole synthase in Escherichia coli is an alphabeta heterodimer of ThiG and ThiH. ThiH is a tyrosine lyase that cleaves the C alpha-C beta bond of tyrosine, generating p-cresol as a by-product, to form dehydroglycine. This reactive intermediate acts as one of three substrates for the thiazole cyclization reaction catalyzed by ThiG. ThiH is a radical S-adenosylmethionine (AdoMet) enzyme that utilizes a [4Fe-4S](+) cluster to reductively cleave AdoMet, forming methionine and a 5'-deoxyadenosyl radical. Analysis of the time-dependent formation of the reaction products 5'-deoxyadenosine (DOA) and p-cresol has demonstrated catalytic behavior of the tyrosine lyase. The kinetics of product formation showed a pre-steady state burst phase, and the involvement of DOA in product inhibition was identified by the addition of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase to activity assays. This hydrolyzed the DOA and changed the rate-determining step but, in addition, substantially increased the uncoupled turnover of AdoMet. Addition of glyoxylate and ammonium inhibited the tyrosine cleavage reaction, but the reductive cleavage of AdoMet continued in an uncoupled manner. Tyrosine analogues were incubated with ThiGH, which showed a strong preference for phenolic substrates. 4-Hydroxyphenylpropionic acid analogues allowed uncoupled AdoMet cleavage but did not result in further reaction (C alpha-C beta bond cleavage). The results of the substrate analogue studies and the product inhibition can be explained by a mechanistic hypothesis involving two reaction pathways, a product-forming pathway and a futile cycle.

Mass-spectrometric and computational study of tryptophan radicals (Trp + H)˙ produced by collisional electron transfer to protonated tryptophan in the gas phase

Hydrogen atom adducts to tryptophan were generated for the first time in the gas phase by collisional electron transfer to protonated tryptophan at 7170 eV kinetic energy. The radicals showed fast dissociations by C(α)-C(β) bond cleavage and cross-ring cleavages occurring on the 7.3 μs time scale. The mechanism of the C(α)-C(β) bond cleavage was explained by ab initio computational analysis of the radical potential energy surface. This showed spontaneous isomerization of the primary tryptophan radical by ammonium hydrogen atom migration to the carboxyl group. The stable intermediate formed by the isomerization can undergo radical-induced scission of the C(α)-C(β) bond in competition with H-atom migrations to the C-2 and C-4 positions of the indole ring. RRKM calculations of unimolecular rate constants on the B3-ROMP2/6-311++G(2d,p) potential energy surface indicated that the C(α)-C(β) bond cleavage was the fastest unimolecular reaction of the radical intermediates within the range of internal energies acquired upon electron transfer. We also report an updated G2(MP2) proton affinity of tryptophan (PA = 946 kJ mol(-1)) and hydrogen atom affinities of the tryptophan indole ring of relevance to electron-based peptide dissociations.

Synthesis of a lignin polymer model consisting of only phenolic β-O-4 linkages and testing its reactivity under alkaline conditions 10th EWLP, Stockholm, Sweden, August 25–28, 2008

Abstract A trimeric β-O-4 lignin model bearing a styrene unit was polymerized in the presence of azobisisobutyronitrile as an initiator of free radical polymerization. The polymer analysis achieved by size exclusion chromatography (SEC), 1H, 13C, 31P NMR, matrix-assisted laser desorption ionization combined with time-of-flight mass spectrometry (MALDI-ToF MS), differential scanning calorimetry, and thermogravimetry indicated its well-defined structure with good thermal stability at temperatures usually applied for alkaline pulping. SEC analysis proved a high degree of polymerization despite the size of the phenolic β-O-4 units in the polymer structure. MALDI-ToF spectrometry indicated that the polymer is composed of phenolic β-O-4 elements only. The chemical reactivity under alkaline conditions of the polymer was studied by both HPLC and 31P NMR. These two methods revealed two main concurrent processes, which include an oxidation of the phenol part followed by Cα-Cβ bond cleavage, and a formation of a quinone methide followed by its β-O-4 bond cleavage.

DFT Calculation on the Stereochemistry of the Allylic Oxidation. Selenium Dioxide-Mediated Oxidation of an Exocyclic Olefinic Hydrindane Compound

Selenium dioxide-mediated oxidation is among the most important transformations for introducing an hydroxy group into the allylic position in the substituted alkenes in organic and biochemical pathways. A unique mode of highly stereoselective interaction of selenium dioxide with olefins, resulting in allylic alcohols, is well exemplified for the preparation of a steroid compound as shown Scheme 1. Since Sharpless and Lauer proposed a mechanism, generally accepted, for this process in 1972 as resulting from an initial ene reaction followed by a [2,3]-sigmatropic rearrangement, there have been considerable efforts on particularly clarifying the stereo-selectivity of this reaction. For example, Singleton and Hang clarified a concerted aspect of the ene step in the allylic oxidation of 2-methyl-2-butene with selenium dioxide (1 → 2 in Scheme 2). Recently we have elucidated the overall pathways in detail by treating theoretically both an ‘ene’ reaction and the 2,3-sigmatropic rearrangement involved in this selenium dioxide-mediated oxidation of 1: the allylic oxidation occurs by complex mechanisms via two competing paths such as anti-approach and syn-approach in the ‘ene’ reaction to produce the (E)allylic alcohol 2, where it has been first noticed the Cα-Cβ bond rotation in 2,3-rearrangement step plays an important part for the stereo-selectivity of the reaction. Now our attention is brought to understand the consequence of the Cα-Cβ bond rotation in the selenic acid intermediate produced from the ene step as to whether it may affects the stereochemical course of the allylic oxidation of a hydrindane compound 5, where an exocyclic olefinic structure is crafted (Scheme 3). Here, we report the results of ab initio studies of the allylic oxidation of 5, focusing on analyzing whether the conformational change via bond rotation in selenic acid intermediate 6 may give rise to produce 9. To study the reaction pathway with relative stabilities for possible transition states during the allylic oxidation of model compound 5 (Scheme 4 and Table 1), we have investigated the energy of possible transition states in SeO2 oxidation of 5 by b3lyp/6-31G* calculation. In ene reaction step, the anti approach of selenium dioxide is energetically favored than the syn one by 1.82 kcal/mol (entry 2 and 3 in Table 1). In the following 1,3-rearrangement step, the O=Segroup approach to the α face of double bond is more favorable than the β face by 2.28 kcal/mol (entry 4 and 5 in Table 1). The compound 7 as a major product should be obtained by the pathway of ts-1a and ts-1a-2α. The other isomer 9 can be formed through the pathway of ts-1a and ts1a-2β, where a Cα-Cβ bond rotation of the ‘ene’ reaction

Mechanisms of photoinduced CαCβ bond breakage in protonated aromatic amino acids

Photoexcitation of protonated aromatic amino acids leads to CαCβ bond breakage among other channels. There are two pathways for the CαCβ bond breakage, one is a slow process (microseconds) that occurs after hydrogen loss from the electronically excited ion, whereas the other is a fast process (nanoseconds). In this paper, a comparative study of the fragmentation of four molecules shows that the presence of the carboxylic acid group is necessary for this fast fragmentation channel to occur. We suggest a mechanism based on light-induced electron transfer from the aromatic ring to the carboxylic acid, followed by a fast internal proton transfer from the ammonium group to the negatively charged carboxylic acid group. The ion formed is a biradical since the aromatic ring is ionized and the carbon of the COOH group has an unpaired electron. Breakage of the weak CαCβ bond gives two even-electron fragments and is expected to quickly occur. The present experimental results together with the ab initio calculations support the interpretation previously proposed.

S-Nitrosylation-induced Conformational Change in Blackfin Tuna Myoglobin

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.