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Significance: Over the past 50 years, the mechanisms for O2 storage and transport have been determined quantitatively on distance scales from millimeters to tenths of nanometers and timescales from seconds to picoseconds. Recent Advances: In this review, I have described four key conclusions from work done by my group and our close colleagues. (i) O2 uptake by mammalian red cells is limited by diffusion through unstirred water layers adjacent to the cell surface and across cell-free layers adjacent to vessel walls. (ii) In most vertebrates, hemoglobins (Hbs) and myoglobins (Mbs), the distal histidine at the E7 helical position donates a strong hydrogen bond to bound O2, which selectively enhances O2 affinity, prevents carbon monoxide poisoning, and markedly slows autoxidation. (iii) O2 binding to mammalian Hbs and Mbs occurs by migration of the ligand through a channel created by upward rotation of the His(E7) side chain, capture in the empty space of the distal pocket, and then coordination with the ferroprotoporphyrin IX (heme) iron atom. (iv) The assembly of Mbs and Hbs occurs by formation of molten globule intermediates, in which the N- and C-terminal helices have almost fully formed secondary structures, but the heme pockets are disordered and followed by high-affinity binding of heme. Critical Issues: These conclusions indicate that there are often compromises between O2 transport function, holoprotein stability, and the efficiency of assembly. Future Directions: However, the biochemical mechanisms underlying these conclusions provide the framework for understanding globin evolution in greater detail and for engineering more efficient and stable globins. [ABSTRACT FROM AUTHOR]

Research indicates that in heme proteins the metal-ligand vibrational frequencies are complex for NO-heme adducts vis a vis their CO-heme counterparts. Furthermore, extra antibonding electron on NO introduce additional complexity. Data indicate that the vibrational frequencies are sensitive to electronic polarization and bond strength to the trans axial ligand.

Antonini and Brunori's 1971 book "Hemoglobin and Myoglobin in Their Reactions with Ligands" was a truly remarkable publication that summarized almost 100 years of research on O 2 binding to these globins. Over the ensuing 50 years, ultra-fast laser photolysis techniques, high-resolution and time resolved X-ray crystallography, molecular dynamics simulations, and libraries of recombinant myoglobin (Mb) and hemoglobin (Hb) variants have provided structural interpretations of O 2 binding to these proteins. The resultant mechanisms provide quantitative descriptions of the stereochemical factors that govern overall affinity, including proximal and distal steric restrictions that affect iron reactivity and favorable positive electrostatic interactions that preferentially stabilize bound O 2. The pathway for O 2 uptake and release by Mb and subunits of Hb has been mapped by screening libraries of site-directed mutants in laser photolysis experiments. O 2 enters mammalian Mb and the α and β subunits of human HbA through a channel created by upward and outward rotation of the distal His at the E7 helical position, is non-covalently captured in the interior of the distal cavity, and then internally forms a bond with the heme Fe(II) atom. O 2 dissociation is governed by disruption of hydrogen bonding interactions with His (E7), breakage of the Fe(II)–O 2 bond, and then competition between rebinding and escape through the E7-gate. The structural features that govern the rates of both the individual steps and overall reactions have been determined and provide the framework for: (1) defining the physiological functions of specific globins and their evolution; (2) understanding the clinical features of hemoglobinopathies; and (3) designing safer and more efficient acellular hemoglobin-based oxygen carriers (HBOCs) for transfusion therapy, organ preservation, and other commercially relevant O 2 transport and storage processes. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated CO migration and binding in CuBMb, a copper-binding myoglobin double mutant (L29H–F43H), by using Fourier transform infrared spectroscopy and flash photolysis over a wide temperature range. This mutant was originally engineered with the aim to mimic the catalytic site of heme–copper oxidases. Comparison of the wild-type protein Mb and CuBMb shows that the copper ion in the distal pocket gives rise to significant effects on ligand binding to the heme iron. In Mb and copper-free CuBMb, primary and secondary ligand docking sites are accessible upon photodissociation. In copper-bound CuBMb, ligands do not migrate to secondary docking sites but rather coordinate to the copper ion. Ligands entering the heme pocket from the outside normally would not be captured efficiently by the tight distal pocket housing the two additional large imidazole rings. Binding at the Cu ion, however, ensures efficient trapping in CuBMb. The Cu ion also restricts the motions of the His64 side chain, which is the entry/exit door for ligand movement into the active site, and this restriction results in enhanced geminate and slow bimolecular CO rebinding. These results support current mechanistic views of ligand binding in hemoglobins and the role of the CuB in the active of heme–copper oxidases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

The unfolding of wild-type holomyoglobin in the ferric State (metMb) appears to be a Simple two- state process, even though hemichrome spectra are often observed and apoMb denaturation involves an intermediate. To resolve these discrepancies, we measured GuHCI-induced, equilibrium unfolding of five sperm whale metMb variants, which were selected to examine the relative importance of apoglobin stability and hemin affinity. Combined analysis of CD, Trp fluorescence, and Soret absorbance titration curves for all the variants requires a Six-State mechanism containing native (N), intermediate (I), and unfolded (U) states of apoMb and their hemin-bound counterparts, NH (holoMb), IH, and UH, respectively. The unfolding parameters for the apoMbs were obtained in independent experiments and then fixed in the analysis of the holoprotein data, where only the affinities of the apoglobin states for hemin were allowed to vary. This cofactor binding analysis applies generally to all globins and led to three specific conclusions. (I) The stability of holo-metMb is determined primarily by the high affinity (Kd 10-3M) of native apoMb (N) for hemin. (2) The partially unfolded intermediate with hemin bound (IH) has a hemichrome spectrum indicative of a bis- histidyl axial coordination and is seen clearly when the stability of the N state or its affinity for hemin is reduced. (3) Although the affinity of the intermediate for hemin (Kdl0-11M) is -` 100-fold lower than that for the native slate, free hemin can bind to it and promote the assembly of the holoprotein. [ABSTRACT FROM AUTHOR]

The FTIR spectra for alkyl isocyanides (CNRs) change from a single VCN band centered at ∼-2175 cm-1 to two peaks at ∼2075 and∼2125cm-1 upon binding to sperm whale myoglobin (Mb). The low- and high-frequency peaks have been assigned to in and Out conformations, respectively. In the in conformation, the ligand is pointing toward the protein interior, and the distal His64(E7) is in a closed position, donates a H-bond to the bound isocyano group, enhances back-bonding, and lowers the C-N bond order. In the out conformation, the ligand side chain points toward solvent through a channel opened by outward rotation of His64. Loss of positive polarity near the binding site causes an increase in C-N bond order. Support for this interpretation is threefold: (I) similar shifts to lower frequency occur for MbCO complexes when H-bond donation from His64(E7) occurs; (2) only one peak at ∼2125 cm-1, indicative of an apolar environment, is observed for CNRs bound to H64A or H64L Mb mutants or to chelated protoheme in soap micelles; and (3) the fraction of in conformation based on FTIR spectra correlates strongly with the fraction of geminate recombination after nanosecond laser photolysis. The in alkyl side chain conformation causes the photodissociated ligand to be "stuck" in the distal pocket, promoting internal rebinding, whereas the out conformation inhibits geminate recombination because part of the ligand is already in an open E7 channel, poised for rapid escape. [ABSTRACT FROM AUTHOR]

A previously undescribed spectrokinetic assay for the entry of water into the distal heme pocket of wild-type and mutant myoglobins is presented. Nanosecond photolysis difference spectra were measured in the visible bands of sperm whale myoglobin as a function of distal pocket mutation and temperature. A small blue shift in the 560-nm deoxy absorption peak marked water entry several hundred nanoseconds after CO photodissociation. The observed rate suggests that water entry is rate-limited by the escape of internal dissociated CO. The heme pocket hydration and geminate recombination yields were found to be the primary factors controlling the overall bimolecular association rate constants for CO binding to the mutants studied. The kinetic analysis provides estimates of 84%. 60%, 40%, 0%, and 99% for the steady-state hydrations of wild-type, H64Q, H64A, H64L, and V68F deoxymyoglobin, respectively. The second-order rate constants for CO and H2O entry into the empty distal pocket of myoglobin are markedly different, 8 × 107 and 2 × 105 M-1·s-1, respectively, suggesting that hydrophobic partitioning of the apolar gas from the aqueous phase into the relatively apolar protein interior lowers the free energy barrier for CO entry. [ABSTRACT FROM AUTHOR]

Compares the properties of wild-type, V68T, and H97D sperm whale myoglobins to determine the relative importance of heme affinity and globin stability on the resistance of the holoprotein to denaturation. Decrease in apoglobin stability due to V68T mutation; Increase in hemin affinity by formation of a strong hydrogen bond between coordinated water and the Thr68 side chain.

Studies the mechanism of hydrogen cyanide binding to myoglobin. Distal amino acid residues; Rate and equilibrium constants for cyanide binding; Equilibrium association constants; Phe46 mutants.

Visible and ultraviolet resonance Raman (RR) spectra are reported for FeIII(NO) adducts of myoglobin variants with altered polarity in the distal heme pockets. The stretching frequencies of the FeIII-NO and N-O bonds, νFeN and νNO are negatively correlated, consistent with backbonding. However, the correlation shifts to lower νNO for variants lacking a distal histidine. DFT modeling reproduces the shifted correlations and shows the shift to be associated with the loss of a lone-pair donor interaction from the distal histidine that selectively strengthens the N-a bond. However, when the model contains strongly electron-withdrawing substituents at the heme β-positions, νFeN and νNO become positively correlated. This effect results from FeIIIN-O bending, which is induced by lone-pair donation to the NNO atom. Other mechanisms for bending are discussed, which likewise lead to a positive νFeN/νNO correlation, including thiolate ligation in heme proteins and electron-donating meso-substituents in heme models. The νFeN/νNO data for the Fe(lll) complexes are reporters of heme pocket polarity and the accessibility of lone pair, Lewis base donors. Implications for biologically important processes, including NO binding, reductive nitrosylation, and NO reduction, are discussed. [ABSTRACT FROM AUTHOR]

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100ps to 3μs. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally “watch” the protein as it executes its function. [Copyright &y& Elsevier]

We have combined Fourier transform infrared/temperature derivative (FTIR-TDS) spectroscopy at cryogenic temperatures and flash photolysis at ambient temperature to examine the effects of polar and bulky amino acid replacements of the highly conserved distal valine 68 in sperm whale myoglobin. In FTIR-TDS experiments, the CO ligand can serve as an internal voltmeter that monitors the local electrostatic field not only at the active site but also at intermediate ligand docking sites. Mutations of residue 68 alter size, shape, and electric field of the distal pocket, especially in the vicinity of the primary docking site (state B). As a consequence, the infrared bands associated with the ligand at site B are shifted. The effect is most pronounced in mutants with large aromatic side chains. Polar side chains (threonine or serine) have only little effect on the peak frequencies. Ligands that migrate toward more remote sites C and D give rise to IR bands with altered frequencies. TDS experiments separate the photoproducts according to their recombination temperatures. The rates and extent of ligand migration among internal cavities at cryogenic temperatures can be used to interpret geminate and bimolecular O[sub 2] and CO recombination at room temperature. The kinetics of geminate recombination can be explained by steric arguments alone, whereas both the polarity and size of the position 68 side chain play major roles in regulating bimolecular ligand binding from the solvent. [ABSTRACT FROM AUTHOR]

Expression levels in animal muscle tissues and in Escherichia coli vary widely for naturally occurring mammalian myoglobins (Mb). To explore this variation, we developed an in vitro transcription and wheat germ extract-based translation assay to examine quantitatively the factors that govern expression of holoMb. We constructed a library of naturally occurring Mbs from two terrestrial and four deep-diving aquatic mammals and three distal histidine mutants designed to enhance apoglobin stability but decrease hemin affinity. A strong linear correlation is observed between cell-free expression levels of holo-metMb variants and their corresponding apoglobin stabilities, which were measured independently by guanidine HCl-induced unfolding titrations using purified proteins. In contrast, there is little dependence of expression on hemin affinity. Our results confirm quantitatively that deep diving mammals have highly stable Mbs that express to higher levels in animal myocytes, E. coli, and the wheat germ cell-free system than Mbs from terrestrial mammals. Our theoretical analyses show that the rate of aggregation of unfolded apoMb is very large, and as a result, the key factor for high level expression of holoMb, and presumably other heme proteins, is an ultra high fraction of folded, native apoglobin that is capable of rapidly binding hemin. This fraction is determined by the overall equilibrium folding constant and not hemin affinity. These results also demonstrate that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability without the need to generate large amounts of protein for in vitro unfolding measurements. [ABSTRACT FROM AUTHOR]

Since the elucidation of the myoglobin (Mb) structure, a histidine residue on the E helix (His-E7) has been proposed to act as a gate with an open or closed conformation controlling access to the active site. Although it is believed that at low pH, the His-E7 gate is in its open conformation, the full relationship between the His-E7 protonation state, its conformation, and ligand migration in Mb is hotly debated. We used molecular dynamics simulations to first address the effect of His-E7 protonation on its conformation. We observed the expected shift from the closed to the open conformation upon protonation, but more importantly, noted a significant difference between the conformations of the two neutral histidine tautomers. We further computed free energy profiles for oxygen migration in each of the possible His-E7 states as well as in two instructive Mb mutants: Ala-E7 and Trp-E7. Our results show that even in the closed conformation, the His-E7 gate does not create a large barrier to oxygen migration and permits oxygen entry with only a small rotation of the imidazole side chain and movement of the E helix. We identify, instead, a hydrophobic site in the E7 channel that can accommodate an apolar diatomic ligand and enhances ligand uptake particularly in the open His-E7 conformation. This rate enhancement is diminished in the closed conformation. Taken together, our results provide a new conceptual framework for the histidine gate hypothesis. [ABSTRACT FROM AUTHOR]

Crystal structures of methyl, ethyl, propyl, and butyl isocyanide bound to sperm whale myoglobin (Mb) reveal two major conformations. In the in conformer, His(E7) is in a "closed" position, forcing the ligand alkyl chain to point inward. In the out conformer, His(E7) is in an "open" position, allowing the ligand side chain to point outward. A progressive increase in the population of the Out conformer is observed with increasing ligand length in P21 crystals of native Mb at pH 7.0. This switch from in to out with increasing ligand size also occurs in solution as measured by the decrease in the relative intensity of the low-frequency (∼2075 cm-1) versus high-frequency (∼2125 cm-1) isocyano bands. In contrast, all four isocyanides in P6 crystals of wild-type recombinant Mb occupy the in conformation. However, mutating either His64 to Ala, creating a "hole" to solvent, or Phe46 to Val, freeing rotation of His64, causes bound butyl isocyanide to point completely outward in P6 crystals. Thus, the unfavorable hindrance caused with crowding a large alkyl side chain into the distal pocket appears to be roughly equal to that for pushing open the His(E7) gate and is easily affected by crystal packing. This structural conclusion supports the "side path" kinetic mechanism for 02 release, in which the dissociated ligand first moves toward the protein interior and then encounters steric resistance, which is roughly equal to that for escaping to solvent through the His(E7) channel. [ABSTRACT FROM AUTHOR]

The major pathway for O2 binding to mammalian myoglobins (Mb) and hemoglobins (Hb) involves transient upward movement of the distal histidine (His-64(E7)), allowing ligand capture in the distal pocket. The mini-globin from Cerebratulus lacteus (CerHb) appears to have an alternative pathway between the E and H helices that is made accessible by loss of the N-terminal A helix. To test this pathway, we examined the effects of changing the size of the E7 gate and closing the end of the apolar channel in CerHb by site-directed mutagenesis. Increasing the size of Gln-44(E7) from Ala to Trp causes variation of association (k'O2) and dissociation (kO2) rate coefficients, but the changes are not systematic. More significantly, the fractions (Fgem 0.05-0.19) and rates (kgem 50-100 µs-1) of geminate CO recombination in the Gln-44(E7) mutants are all similar. In contrast, blocking the entrance to the apolar channel by increasing the size of Ala-55(E18) to Phe and Trp causes the following: 1) both k'O2 and kO2 to decrease roughly 4-fold; 2) Fgem for CO to increase from ∼0.05 to 0.45; and 3) kgemn to decrease from ∼80 to ∼9 µs-1,as ligands become trapped in the channel. Crystal structures and low temperature Fourier-transform infrared spectra of Phe-55 and Trp-55 CerHb confirm that the aromatic side chains block the channel entrance, with little effect on the distal pocket. These results provide unambiguous experimental proof that diatomic ligands can enter and exit a globin through an interior channel in preference to the more direct E7 pathway. [ABSTRACT FROM AUTHOR]

Distantly related members of the hemoglobin (Hb) superfamily including red blood cell Hb, muscle myoglobin (Mb) and the microbial flavohemoglobin (flavoHb) dioxygenate nitric oxide (•NO). The reaction serves important roles in •NO metabolism and detoxification throughout the aerobic biosphere. Analysis of the stoichiometric product nitrate shows greater than 99% double O-atom incorporation from Hb18O2, Mb18O2 and flavoHb18O2 demonstrating a conserved high fidelity •NO dioxygenation mechanism. Whereas, reactions of •NO with the structurally unrelated Turbo cornutus MbO2 or free superoxide radical (-O•2) yield sub-stoichiometric nitrate showing low fidelity O-atom incorporation. These and other results support a •NO dioxygenation mechanism involving (1) rapid reaction of •NO with FeIII−-O•2 a intermediate to form FeIII−OONO and (2) rapid isomerization of the FeIII−OONO intermediate to form nitrate. A sub-microsecond isomerization event is hypothesized in which the O–O bond homolyzes to form a protein caged [FeIV=O•NO2] intermediate and ferryl oxygen attacks •NO2 to form nitrate. Hb functions as a •NO dioxygenase by controlling O2 binding and electrochemistry, guiding •NO diffusion and reaction, and shielding highly reactive intermediates from solvent water and biomolecules. [Copyright &y& Elsevier]

The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. Below 200 K, the transition-state enthalpy barrier associated with the fastest (∼10 ps) recombination phase is found to be zero and a slower geminate phase (∼200 ps) reveals a small enthalpic barrier (∼3 ± 1 kJ/mol). Both of the kinetic rates slow slightly in the myoglobin (Mb) samples above 200 K, suggesting that a small amount of protein relaxation takes place above the solvent glass transition. When the temperature dependence of the NO recombination in Mb is studied under conditions where the distal pocket is mutated (e.g., V68W), the rebinding kinetics lack the slow phase. This is consistent with a mechanism where the slower (∼200 ps) kinetic phase involves transitions of the NO ligand into the distal heme pocket from a more distant site (e.g., in or near the Xe4 cavity). Comparison of the temperature- dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol reveals that the fast (enthalpically barrierless) NO rebinding process observed below 200 K is independent of the presence or absence of the proximal histidine ligand. In contrast, the slowing of the kinetic rates above 200 K in MbNO disappears in the absence of the protein. Generally, the data indicate that! in contrast to CO, the NO ligand binds to the heme iron through a "harpoon" mechanism where the heme iron out-of-plane conformation presents a negligible enthalpic barrier to NO rebinding. These observations strongly support a previous analysis (Srajer et al. J. Am. Chem. Soc. 1988, 110, 6656-6670) that primarily attributes the low-temperature stretched exponential rebinding of MbCO to a quenched distribution of heme geometries. A simple model, consistent with this prior analysis, is presented that explains a variety of MbNO rebinding experiments, including the dependence of the kinetic amplitudes on the pump photon energy. [ABSTRACT FROM AUTHOR]

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 ↵ 65 °C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B ↵ A, C ↵ A, and D ↵ A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B ↵ A and C ↵ A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly. [ABSTRACT FROM AUTHOR]

HmuO, a heine oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 μM-1 s-1 and 0.22 s-1, respectively, yielding an 02 affinity of 21 μM-l, which is -20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3–4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor 02 binding. Resonance Raman spectra show that the Fe—O2 vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe—O&mdsh;O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe—O—O angle is 110°, the O—O bond is pointed toward the heme α-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound 02 and the amide nitro. gen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O—O bond is orthogonal to the plane [ABSTRACT FROM AUTHOR]

We report picosecond time-resolved x-ray diffraction from the myoglobin (Mb) mutant in which Leu[sup29] is replaced by Phe (L29F mutant). The frame-by-frame structural evolution, resolved to 1.8 angstroms, allows one to literally "watch" the protein as it executes its function. Time-resolved mid-infrared spectroscopy of flash-photolyzed L29F MbCO revealed a short-lived CO intermediate whose 140-ps lifetime is shorter than that found in wild-type protein by a factor of 1000. The electron density maps of the protein unveil transient conformational changes far more dramatic than the structural differences between the carboxy and deoxy states and depict the correlated side-chain motion responsible for rapidly sweeping CO away from its primary docking site. [ABSTRACT FROM AUTHOR]