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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 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]

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]

We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100ps to 10μs. The proximity of the B site in the β subunit is about 0.25Å closer to its A binding site, and its kBA rebinding rate (∼300μs−1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2. [ABSTRACT FROM AUTHOR]

α-Hemoglobin stabilizing protein (AHSP) is a molecular chaperone that binds monomeric α-subunits of human hemoglobinA( HbA) and modulates heme iron oxidation and subunit folding states. Although AHSPβαHb complexes autoxidize more rapidly than HbA, the redox mechanisms appear to be similar. BothmetHbAand isolated met-β-subunits undergo further oxidation in the presence of hydrogen peroxide (H2O2) to form ferryl heme species. Surprisingly, much lower levels of H2O2- induced ferryl heme are produced by free met-α-subunits as compared with met-β-subunits, and no ferryl heme is detected in H2O2-treated AHSPβmet-α-complex at pH values from 5.0 to 9.0 at 23 °C. Ferryl heme species were similarly not detected in AHSPβmet-α Pro-30 mutants known to exhibit different rates of autoxidation and hemin loss. EPRdata suggest that protein-based radicals associated with the ferryl oxidation state exist within HbA α- and β-subunits. In contrast, treatment of free α-subunits with H2O2 yields much smaller radical signals, and no radicals are detected when H2O2 is added to AHSPβα-complexes. AHSP binding also dramatically reduces the redox potential of α-subunits, from+40 to-78mVin 1 M glycine buffer, pH 6.0, at 8 °C, demonstrating independently that AHSP has a much higher affinity for Fe(III) versus Fe(II) α-subunits. Hexacoordination in the AHSPβmet-α complex markedly decreases the rate of the initial H2O2 reaction with iron and thus provides α-subunits protection against damaging oxidative reactions. [ABSTRACT FROM AUTHOR]

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 ≈ 200–600 s-1) and moderate O2 affinity (KO2 ≈1 μM-1) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 °C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 → Val CerHbO2 at 1.70 Å resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 Å). The dramatic functional and spectral effects of the Thr-E11 → Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val ‘frees’ the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm-1 decrease in Vco of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites. [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]

The ligand binding properties and resistances to denaturation of >300 different site-directed mutants of sperm whale, pig, and human myoglobin have been examined over the past 15 years. This library of recombinant proteins has been used to derive chemical mechanisms for ligand binding and to examine the factors governing holo- and apoglobin stability. We have also examined the effects of mutagenesis on the dioxygenation of NO by MbO2 to form NO3− and metMb. This reaction rapidly detoxifies NO and is a key physiological function of both myoglobins and hemoglobins. The mechanisms derived for O2 binding and NO dioxygenation have been used to design safer, more efficient, and more stable heme protein-prototypes for use as O2 delivery pharmaceuticals in transfusion therapy (i.e. blood substitutes). An interactive database is being developed (http://olsonnt1.bioc.rice.edu/web/myoglobinhome.asp) to allow rapid access to the ligand binding parameters, stability properties, and crystal structures of the entire set of recombinant myoglobins. The long-range goal is to use this library for developing general protein engineering principles and for designing individual heme proteins for specific pharmacological and industrial uses. [Copyright &y& Elsevier]