Showing papers on "Myoglobin published in 1997"

Myoglobin discriminates between O2, NO, and CO by electrostatic interactions with the bound ligand

Abstract: Most biological substrates have distinctive sizes, shapes, and charge distributions which can be recognized specifically by proteins. In contrast, myoglobin must discriminate between the diatomic gases O2, CO, and NO which are apolar and virtually the same size. Selectivity occurs at the level of the covalent Fe-ligand complexes, which exhibit markedly different bond strengths and electrostatic properties. By pulling a water molecule into the distal pocket, His64(E7)1 inhibits the binding of all three ligands by a factor of ∼10 compared to that observed for protoheme-imidazole complexes in organic solvents. In the case of O2 binding, this unfavorable effect is overcome by the formation of a strong hydrogen bond between His64(E7) and the highly polar FeO2 complex. This favorable electrostatic interaction stabilizes the bound O2 by a factor of ∼1000, and the net result is a 100-fold increase in overall affinity compared to model hemes or mutants with an apolar residue at position 64. Electrostatic interaction between FeCO and His64 is very weak, resulting in only a two- to three-fold stabilization of the bound state. In this case, the inhibitory effect of distal pocket water dominates, and a net fivefold reduction in K CO is observed for the wild-type protein compared to mutants with an apolar residue at position 64. Bound NO is stabilized ∼tenfold by hydrogen bonding to His64. This favorable interaction with FeNO exactly compensates for the tenfold inhibition due to the presence of distal pocket water, and the net result is little change in K NO when the distal histidine is replaced with apolar residues. Thus, it is the polarity of His64 which allows discrimination between the diatomic gases. Direct steric hindrance by this residue plays a minor role as judged by: (1) the independence of K O2, K CO, and K NO on the size of apolar residues inserted at position 64, and (2) the observation of small decreases, not increases, in CO affinity when the mobility of the His64 side chain is increased. Val68(E11) does appear to hinder selectively the binding of CO. However, the extent is no more than a factor of 2–5, and much smaller than electrostatic stabilization of bound O2 by the distal histidine.

Proton-coupled electron transfer from electrodes to myoglobin in ordered biomembrane-like films

Abstract: Voltammetry and visible and infrared spectroscopy were used to explore protonation equilibria coupled to electron transfer between electrodes and the heme protein myoglobin (Mb) in thin liquid crys...

Ultrafast rotation and trapping of carbon monoxide dissociated from myoglobin

Abstract: The nature of ligand motion within proteins has been investigated by measuring femtosecond time-resolved infrared (IR) spectra of CO photodissociated from the haem of myoglobin. Upon dissociation, the CO rotates approximately 90 degrees and becomes trapped within a ligand docking site located near the binding site. Two trajectories, distinguished spectroscopically and kinetically with time constants of 0.20 +/- 0.05 ps and 0.52 +/- 0.10 ps, lead to CO located within the docking site with opposite orientations. The protein reorganizes about the "docked' CO with a time constant of 1.6 +/- 0.3 ps and quickly establishes an energetic barrier that inhibits the reverse rebinding process.

Serum concentrations of myoglobin vs human heart-type cytoplasmic fatty acid-binding protein in early detection of acute myocardial infarction

Abstract: We compared the diagnostic utility of serum concentrations of human heart-type cytoplasmic fatty acid-binding protein (H-FABPc), myoglobin, and their ratio for the early diagnosis of acute myocardial infarction (AMI) in 104 healthy volunteers and 165 patients at admission within 6 h of the onset of chest pain. The ROC curves of the H-FABPc [0.946, 95% confidence interval (CI) = 0.913–0.979] and myoglobin (0.895, 95% CI = 0.846–0.944) between patients with AMI and healthy volunteers were significantly greater than the area under the ratio of myoglobin to H-FABPc (0.823, 95% CI = 0.765–0.881). In 165 patients, the sensitivity (81.8%, 95% CI = 74.2–89.4%), specificity (86.4%, 95% CI = 78.1–94.6%), and predictive accuracy (83.6%, 95% CI = 78.0–89.3%) of H-FABPc >12 μg/L in diagnosing AMI were significantly higher than those of myoglobin, and were similar to those of the combination of H-FABPc >12 μg/L and the ratio ≤14. We conclude that H-FABPc is a more sensitive and specific marker than myoglobin for the early diagnosis of AMI, and that their ratio cannot give a clear advantage over the measurement of H-FABPc alone.

Functional analogs of heme protein active sites

Abstract: This article summarizes my research in biomimetic chemistry on the design and study of functional models for the active sites of metalloproteins such as hemoglobin, myoglobin, and cytochrome c oxid...

Identification of conformational substates involved in nitric oxide binding to ferric and ferrous myoglobin through difference Fourier transform infrared spectroscopy (FTIR).

Abstract: Hemeproteins play an important role in the signaling processes mediated by nitric oxide (NO). For example, the production of NO by nitric oxide synthase, the activation of guanylate cyclase by binding NO, and the scavenging of NO by hemoglobin, myoglobin, and cytochrome c oxidase all occur through unique mechanisms of interaction between NO and hemeproteins. Unlike carbon monoxide (CO) and oxygen (O2), which have been studied extensively, the reactions of NO with ferric and ferrous hemeproteins are not as well characterized. In this work, NO binding to myoglobin is studied using cryogenic optical spectroscopy and Fourier transform infrared spectroscopy (FTIR) in order to characterize the ligand-bound and photoproduct states involved in the interaction of NO with the heme iron and the distal pocket of the protein. For ferrous nitrosyl myoglobin (MbIINO), optical spectroscopy is used to show that the ligand-bound state can be converted to >95% stable photoproduct below 10 K. The Soret peak of the photoproduct is red-shifted by 4 nm relative to deoxy-myoglobin (Mb), similar to previous results for carbonmonoxy- (MbCO) and oxy-myoglobin (MbO2) (Miller et al., 1996). MbIINO completely rebinds by 35 K, indicating that the rebinding barrier for NO is lower than MbCO, consistent with room temperature picosecond kinetic measurements. For ferric nitrosyl myoglobin (MbIIINO), we find that the photoproduct yield at cryogenic temperatures is less than unity and dependent on the distal pocket residue. Native MbIIINO has a lower photoproduct yield than the mutant, MbIII(H64L)NO, where the distal histidine is replaced by leucine. The rebinding rates for the native and mutant species are similar to each other and to MbIINO. By using FTIR difference spectroscopy (photolyzed/unphotolyzed) of isotopically labeled ferrous nitrosyl myoglobin (MbIINO), the NO stretching frequencies in both the ligand-bound states and photoproduct states are determined. Two ligand-bound conformational states (1607 and 1613 cm-1) and two photoproduct conformational states (1852 and 1857 cm-1) are observed for MbIINO. This is the first direct observation of photolyzed NO in the distal pocket of myoglobin. The ligand-bound frequencies are consistent with a bent MbIINO moiety, where the unpaired pi*(NO) electron remains localized on NO, causing nu(N-O) to be approximately 300 cm-1 lower than MbIIINO. Similar to MbO2, we suggest that Nepsilon of the distal histidine is protonated, forming a hydrogen bond to the NO ligand. For native MbIIINO, a single ligand-bound conformational state with respect to nu(N-O) is observed at 1927 cm-1. This frequency decreases to 1904 cm-1 for the mutant, MbIII(H64L)NO, contrary to the increase of the carbon monoxide (CO) stretching frequency in the isoelectronic MbII(H64L)CO mutant versus native MbCO. For linear MbIIINO, we suggest that backbonding from the unpaired pi*(NO) electron to iron results in an increased positive charge on the NO ligand, Fe(delta-)-NO(delta+). This can be facilitated by tautomerism of the distal histidine, leaving Nepsilon of the imidazole ring unprotonated and able to accept positive charge from the Fe(delta-)-NO(delta+) moiety, resulting in a higher bond order (and a 23 cm-1 shift to higher frequency) for native MbIIINO versus MbIII(H64L)NO, where this interaction is absent. These different interactions between the distal histidine and the ferrous versus ferric species illustrate potential ways the protein can stabilize the bound ligand and demonstrate the versatile nature by which NO can bind to hemeproteins.

Vibrational frequency shifts as a probe of hydrogen bonds: thermal expansion and glass transition of myoglobin in mixed solvents

Abstract: The contribution of hydrogen bonds to protein-solvent interactions and their impact on structural flexibility and dynamics of myoglobin are discussed. The shift of vibrational peak frequencies with the temperature of myoglobin in sucrose/water and glycerol/water solutions is used to probe the expansion of the hydrogen bond network. We observe a characteristic change in the temperature slope of the O-H stretching frequency at the glass transition which correlates with the discontinuity of the thermal expansion coefficient. The temperature-difference spectra of the amide bands show the same tendency, indicating that stronger hydrogen bonding in the bulk affects the mainchain solvent interactions in parallel. However, the hydrogen bond strength decreases relative to the bulk solvent with increasing cosolvent concentration near the protein surface, which suggests preferential hydration. Weaker and/or fewer hydrogen bonds are observed at low degrees of hydration. The central O-H stretching frequency of protein hydration water is red-shifted by 40 cm-1 relative to the bulk. The shift increases towards lower temperatures, consistent with contraction and increasing strength of the protein-water bonds. The temperature slope shows a discontinuity near 180 K. The contraction of the network has reached a critical limit which leads to frozen-in structures. This effect may represent the molecular mechanism underlying the dynamic transition observed for the mean square displacements of the protein atoms and the heme iron of myoglobin.

Folding propensities of peptide fragments of myoglobin

Abstract: Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.

Real-time in-vivo measurement of myoglobin oxygen saturation

Abstract: A method for determining myoglobin oxygen fractional saturation in vivo in muscle tissue and intracellular oxygen tension (pO 2 ), typically in the presence of hemoglobin, is provided. The method comprises measuring the absorption spectrum of the tissue using spectrographic equipment known to the art, including equipment for non-invasively taking spectroscopic measurements of tissue, or adaptations of such equipment to provide the preferred measurements described herein, or using spectrographic equipment specifically designed as described herein for use in non-invasive measurements of reflectance spectra. The measured spectrum is corrected for light scattering effects, such as by taking the second derivative of the data, or by using other means which are known to the art. Myoglobin fractional oxygen saturation is calculated from the measured spectrum employing calibration coefficients that are themselves calculated from application of multivariate analysis to a calibration set created from the second derivatives of absorption spectra. The calibration set spectra preferably representing absorbances of (1) a range of concentrations of hemoglobin (wherein a range of concentrations of oxyhemoglobin and deoxyhemoglobin are present), (2) one concentration of myoglobin (preferably selected to match the concentration of myoglobin in the target muscle tissue) with varying relative amounts of oxy- and deoxymyoglobin and (3) a range of concentrations of scattering agents to mimic scattering encountered in target tissue. The range of concentrations of oxy- and deoxyhemoglobin and the relative amounts of oxy- and deoxymyoglobin represented in calibration set spectra span the range of concentrations of these species encountered in the target tissue. Sample tissue spectra are preferably measured in the visible, the near-infrared or both wavelength ranges. Diffuse reflectance spectroscopy is the preferred method for obtaining spectral data. A partial least squares (PLS) method is preferably used to calculate calibration coefficients from the calibration set which in turn are used to calculate myoglobin oxygen saturation from the measured data. Other means known to the art may also be used. Measured myoglobin oxygen saturation determinations can be used to calculate intracellular oxygen tension, if accurate p50 values at appropriate physiologic pH and temperature for myoglobin-oxygen dissociation are available. An improved method of accurate determination of myoglobin-oxygen dissociation curves under physiologically relevant conditions is also provided.

Dissociation of heme from myoglobin and cytochrome b5: comparison of behavior in solution and the gas phase.

Abstract: The relationship of the structure of a protein in solution to the structure of a gas-phase protein ion and the manner in which gas-phase protein ions bind small molecules noncovalently are topics of current debate. To address these issues, the stability of heme binding to wild-type and variant forms of apomyoglobin and apocytochrome b5 has been studied in the gas phase by electrospray mass spectrometry (ES-MS) and compared with the stability of heme binding to the same proteins in solution. The voltage required to dissociate ions of the heme−protein complexes in the orifice−skimmer region of an electrospray mass spectrometer, a measure of the complex stability, is found to be correlated with the activation energy for dissociation of the complexes in solution across a series of proteins in which the number of hydrogen bonds between the heme propionate groups and surface residues is systematically reduced. However, variants in which the hydrogen bonds to the proximal histidine have been removed are destabil...

A comparison between molecular dynamics and X-ray results for dissociated CO in myoglobin.

Abstract: The distribution of carbon monoxide after photodissociation in the myoglobin haem pocket has been investigated using molecular dynamics simulations at 300 K. The results show that both intermediates (one close to the haem iron and one further away) observed in recent low temperature X-ray studies of photodissociated CO have a high probability of occurrence, even at ambient temperatures. The fact that the O of CO is oriented toward the haem iron in the closer intermediate provides an explanation for the slow rate of CO geminate rebinding. A refinement against X-ray data generated from the molecular dynamics simulations indicates that the CO has a broader distribution in the haem pocket than is apparent from the experimental electron density. This effect is likely to be general for systems containing highly mobile groups.

Electrochemical Generation and Reactions of Ferrylmyoglobins in Water and Microemulsions

Abstract: Ferrylmyoglobin species, which are active oxidant forms of the protein myoglobin, were obtained by electrochemical reduction of metmyoglobin [MbFeIII] in the presence of oxygen in aqueous neutral buffer and in microemulsions of oil, water, and cationic surfactant. Reduction of myoglobin at −0.4 V vs SCE catalyzed the reduction of oxygen to hydrogen peroxide at the electrode. Hydrogen peroxide oxidizes metmyoglobin in solution to give the radical ferrylmyoglobin •X−MbFeIVO, which is known to decay rapidly to the non-radical MbFeIVO. This complex reduction−oxidation process converted nearly all of the 30 μM myoglobin in a spectroelectrochemical cell to ferrylmyoglobins in 15 min in pH 7.3 buffer and in 18 min in microemulsions. Characteristic ferryl heme absorbance bands near 421, 548, and 584 nm were used to identify products. Confirmation of ferrylmyoglobins was provided by reductions to metmyoglobin with ascorbate and by myoglobin-mediated electrochemical epoxidation of styrene. Fiftyfold higher yields o...

Estimation of myocardial infarct size from plasma myoglobin or fatty acid-binding protein. Influence of renal function.

Abstract: Myoglobin (M(r) 18,000) and fatty acid-binding protein (M(r) 15,000), are low molecular mass cytoplasmic proteins that are considered useful biochemical markers for early detection or exclusion of acute myocardial infarction, and also for early estimation of infarct size. As each of these proteins shows renal clearance, we studied the influence of renal function on the estimation of infarct size from their plasma concentration curves. For this, infarct size estimated from plasma myoglobin or fatty acid-binding protein release curves was compared with that estimated with the established infarct size markers hydroxybutyrate dehydrogenase and creatine kinase, which are not influenced by changes in renal function. The discordance between infarct size estimates was related to renal function. Creatine kinase (EC 2.7.3.2), hydroxybutyrate dehydrogenase (EC 1.1.1.27), myoglobin, fatty acid-binding protein and creatinine were assayed serially in plasma samples obtained frequently and for at least 72 hours after the start of thrombolytic therapy in 20 patients with acute myocardial infarction. Cumulative release of the different cardiac markers was calculated by using a two-compartment model for circulating proteins. Mean tissue contents of 156 U/g for hydroxybutyrate dehydrogenase, 2163 U/g for creatine kinase, 2.79 mg/g for myoglobin and 0.57 mg/g wet weight for fatty acid-binding protein, were used to express infarct size in gram-equivalents of healthy myocardium per litre of plasma (g-eq/l). Mean plasma creatinine was obtained by averaging the creatinine concentrations measured in all plasma samples taken during the first 24 hours after acute myocardial infarction. A relation was found between the mean plasma creatinine concentration during the first 24 hours after acute myocardial infarction and the discordance between infarct size estimated from cumulative hydroxybutyrate dehydrogenase release, compared to infarct size estimated from cumulative myoglobin or fatty acid-binding protein release. For patients with mean plasma creatinine concentrations within the reference interval for creatinine (group 1, n = 15) a good agreement was found between infarct size estimated from myoglobin or fatty acid-binding protein plasma curves and that estimated with either hydroxybutyrate dehydrogenase or creatine kinase. However, for patients with a mean creatinine concentration above the upper reference limit (group 2, n = 5), infarct size calculated from plasma myoglobin or fatty acid-binding protein release curves was markedly overestimated, especially for larger infarcts. Estimation of infarct size from serial plasma myoglobin or fatty acid-binding protein concentrations is possible in the first 24 hours after the onset of symptoms, but only in patients with normal renal function, as estimated from plasma creatinine concentrations.

Iron L-Edge X-ray Absorption Spectroscopy of Myoglobin Complexes and Photolysis Products

Abstract: We demonstrate the first application of L-edge X-ray absorption spectroscopy (XAS) to the electronic characterization of biological photolysis products. The experimental L-edge XAS spectra of deoxymyoglobin (deoxy Mb), oxymyoglobin (MbO2), carbonmonoxymyoglobin (MbCO), and the low-temperature photoproducts (Mb*CO and Mb*O2) are presented and compared to simulated spectra using a ligand field multiplet calculation. This analysis indicates that MbCO and MbO2 are both low spin and does not support some previous studies which suggest that MbO2 has an intermediate spin. Both photoproducts, Mb*CO and Mb*O2, are different from deoxy Mb in the FeII electronic structure. In addition, different low-temperature photolysis intermediates are suggested for MbCO and MbO2. The L-edge XAS spectra for FeIII in aquometmyoglobin (met Mb) and azidomet myoglobin (MbN3) provide a comparison of the ferrous versus ferric myoglobin species. Finally, the special advantages of using soft X-ray absorption spectroscopy for understandi...

Aza-Crown-Capped Porphyrin Models of Myoglobin: Studies of the Steric Interactions of Gas Binding

Abstract: A series of myoglobin active site analogues (1−6) has been synthesized and characterized. These synthetic models differ in their cavity dimensions, and have been designed to demonstrate the effects of steric factors on O2 and CO binding affinities. Quantitative gas titrations were employed to measure these affinities, yielding M values that are strikingly lower than those reported for hemoglobin and myoglobin. The 1,4,7-triazacyclononane-capped porphyrin 1 has about 1200 times the CO affinity but only about 10 times the O2 affinity of the cyclam-capped porphyrin 2, suggesting a more open gas binding cavity for 1. The cavity dimensions and conformation of 2 were determined by single-crystal X-ray structural analysis of the Zn analogue 7. This paper unequivocally demonstrates that steric effects can control the ratio of O2/CO binding constants.

Protein Engineering Strategies for Designing More Stable Hemoglobin-based Blood Substitutes

Abstract: Over the past five years our laboratory has been using rational, comparative, and random combinatorial mutagenesis strategies to optimize the alpha and beta subunits of recombinant human hemoglobin (Hb) for efficient O2 transport, greater stability, and minimum interference with vascular activity. In each approach, mammalian myoglobin (Mb) has been used as a prototype to develop experimental methodologies and to study the stereochemical mechanisms that govern O2 affinity, discrimination against CO, rates of ligand binding, auto- and chemically induced oxidation, resistance to hemin loss, and stability to globin denaturation. Multiple replacements in the distal portion of the heme pocket have been designed rationally to lower oxygen affinity and at the same time inhibit oxidative side reactions. The P50 values are adjusted by altering electrostatic and steric interactions between the bound ligand and residues at the Leu(B10), His(E7), and Va(E11) positions. Large apolar residues (Leu, Phe, Trp) at the B10 and E11 positions inhibit NO-induced and autooxidation in both myoglobin and hemoglobin by excluding oxidants and proton donors from the immediate vicinity of the bound ligand. Similar strategies appear to have evolved in a number of animal myoglobins and hemoglobins which have unusual amino acids at the E7, B10, and E11 positions. Random combinatorial mutagenesis techniques have been developed to insert new amino acid combinations near the bound ligand in sperm whale Mb. The objective is to obtain "unnatural" distal pocket structures that enhance O2 transport and resistance to oxidation by alternative mechanisms.

Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket.

Abstract: The reaction of hydrogen peroxide with a number of variants of sperm-whale myoglobin in which the distal pocket histidine residue (His64) had been mutated was studied with a combination of stopped-flow spectroscopy and freeze-quench EPR. The rate of the initial bimolecular reaction with hydrogen peroxide in all the proteins studied was found to depend on the polarity of the amino acid side chain at position 64. In wild-type myoglobin there were no significant optical changes subsequent to this reaction, suggesting the rapid formation of the well-characterized oxyferryl species. This conclusion was supported by freeze-quench EPR data, which were consistent with the pattern of reactivity previously reported [King and Winfield (1963) J. Biol. Chem. 238, 1520-1528]. In those myoglobins bearing a mutation at position 64, the initial bimolecular reaction with hydrogen peroxide yielded an intermediate species that subsequently decayed via a second hydrogen peroxide-dependent step leading to modification or destruction of the haem. In the mutant His64-->Gln the calculated electronic absorption spectrum of the intermediate was not that of an oxyferryl species but seemed to be that of a low-spin ferric haem. Freeze-quench EPR studies of this mutant and the apolar mutant (His64-->Val) revealed the accumulation of a novel intermediate after the first hydrogen peroxide-dependent reaction. The unusual EPR characteristics of this species are provisionally assigned to a low-spin ferric haem with bound peroxide as the distal ligand. These results are interpreted in terms of a reaction scheme in which the polarity of the distal pocket governs the rate of binding of hydrogen peroxide to the haem iron and the residue at position 64 governs both the rate of heterolytic oxygen scission and the stability of the oxyferryl product.

Assessment of Coronary Reperfusion After Thrombolysis With a Model Combining Myoglobin, Creatine Kinase–MB, and Clinical Variables

Abstract: Background Several biochemical markers have been investigated for the noninvasive assessment of reperfusion after myocardial infarction. Because myoglobin is released very soon after myocardial injury and clears rapidly after reperfusion, it may prove to be an excellent marker of occlusion and reperfusion. Methods and Results We examined the relation between various myoglobin measures and Thrombolysis In Myocardial Infarction (TIMI) flow grade in 96 patients enrolled in a study of front-loaded thrombolysis who underwent 90-minute angiography. We also combined myoglobin measures with models that include clinical and creatine kinase–MB variables. The myoglobin level measured within 10 minutes of acute angiography showed the best overall performance and was used for later analyses. Of the clinical variables examined, only time from symptom onset to thrombolysis and chest pain grade at angiography discriminated among TIMI flow grades. Combining the 90-minute myoglobin level and these clinical variables showed...

Direct Observation of Nitrosylated Heme in Myoglobin and Hemoglobin by Electrospray Ionization Mass Spectrometry

Abstract: Using electrospray ionization mass spectrometry (ESI-MS), we demonstrate the direct observation of NO attached to the heme moiety in horse heart myoglobin (Mb) and in the α- and β-chains of human hemoglobin (Hb). It was found that a narrow range of ESI-MS conditions conspire to make observation of Fe−NO interactions challenging, and this is presumably the reason why earlier attempts by other research groups to detect intact Fe−NO products by mass spectrometry were unsuccessful. For Mb and Hb, mass shifts are observed that are consistent with NO modification of the hemoproteins. ESI mass spectra of the apoprotein portions of Mb and Hb in the presence of NO demonstrated the absence of NO modification of the polypeptide backbones. UV/vis spectra of both Mb/NO and Hb/NO solutions, recorded at the time of ESI-MS analysis, demonstrated hemoproteinII−NO formation. To test the hypothesis that intact nitrosylated heme groups are observable by ESI-MS, a nitrosylated model metalloporphyrin was studied. The ESI mass ...