Biomedical subjects

John S Olson

Publications and source records attributed to John S Olson.

At least 19 recordsLinked to original sources

PubMed2006

Biochemical fates of alpha hemoglobin bound to alpha hemoglobin-stabilizing protein AHSP.

Alpha hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds free alpha hemoglobin (alphaHb) to maintain its structure and limit its pro-oxidant activity. Prior studies have defined two different alphaHb.AHSP complexes. Binding of AHSP to Fe(II) alphaHb induces an unusual configuration in which the F helix of the globin becomes disordered and the heme ring becomes solvent-exposed. Over time, this intermediate oxidizes to form a stable hemichrome in which the proximal (F8) and distal (E7) histidines are coordinated to the heme iron atom. The addition of betaHb to either Fe(II) or Fe(III) alphaHb.AHSP displaces AHSP to generate tetrameric (alpha(2)beta(2)) HbA species. The biochemical properties and in vivo significance of the two alphaHb.AHSP complexes are poorly understood. Here we show that Fe(III) alphaHb.AHSP forms from auto-oxidation of oxygenated alphaHb bound to AHSP and that this process is greatly accelerated at physiologic temperature and oxygen pressures. In contrast to free Fe(III) alphaHb hemichromes, AHSP-bound Fe(III) alphaHb does not precipitate and can be recycled into functional HbA. This requires enzymatic reduction of AHSP-bound alphaHb, either prior to or after extraction by beta subunits. In contrast, reaction of Fe(II) alphaHb-AHSP with betaHb generates functional HbA directly. Our findings support a model in which AHSP can either stabilize alphaHb transiently en route to HbA formation during normal erythropoiesis or convert excessive free alphaHb into a more chemically inert state from which recovery of alphaHb is possible by redox cycling.

Binding Sites↗

PubMed2006

The mechanism of direct heme transfer from the streptococcal cell surface protein Shp to HtsA of the HtsABC transporter.

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.

Adenosine Triphosphate↗

PubMed2006

Hemoglobins dioxygenate nitric oxide with high fidelity.

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 Hb(18)O(2), Mb(18)O(2) and flavoHb(18)O(2) demonstrating a conserved high fidelity .NO dioxygenation mechanism. Whereas, reactions of .NO with the structurally unrelated Turbo cornutus MbO(2) 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 a Fe(III-)O.(2) intermediate to form Fe(III-)OONO and (2) rapid isomerization of the Fe(III-)OONO intermediate to form nitrate. A sub-microsecond isomerization event is hypothesized in which the O-O bond homolyzes to form a protein caged [Fe(IV)O .NO(2)] intermediate and ferryl oxygen attacks .NO(2) to form nitrate. Hb functions as a .NO dioxygenase by controlling O(2) binding and electrochemistry, guiding .NO diffusion and reaction, and shielding highly reactive intermediates from solvent water and biomolecules.

Animals↗

PubMed2006

Water and ligand entry in myoglobin: assessing the speed and extent of heme pocket hydration after CO photodissociation.

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 H(2)O entry into the empty distal pocket of myoglobin are markedly different, 8 x 10(7) and 2 x 10(5) 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.

Animals↗

PubMed2005

Temperature-dependent studies of NO recombination to heme and heme proteins.

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 (approximately 10 ps) recombination phase is found to be zero and a slower geminate phase (approximately 200 ps) reveals a small enthalpic barrier (approximately 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 (approximately 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.

Animals↗

PubMed2005

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities.

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 degrees 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.

Animals↗

PubMed2005

Thermoglobin, oxygen-avid hemoglobin in a bacterial hyperthermophile.

The hemoglobin family of proteins, ubiquitous in all domains of life, evolved from an ancestral protein of primordial function to extant hemoglobins that perform a myriad of functions with diverged biochemical properties. Study of homologs in bacterial hyperthermophiles may shed light on both mechanisms of adaptation to extreme conditions and the nature of the ancestral protein. A hemoglobin was identified in Aquifex aeolicus, cloned, recombinantly expressed, purified, and characterized. This hemoglobin is monomeric, resistant to thermal and chemical denaturation, pentacoordinate in the ferrous deoxygenated state, and oxygen-avid. The oxygen equilibrium dissociation constant is approximately 1 nm at room temperature, due in part to a hydrogen bond between the bound ligand and a tyrosine residue in the distal pocket. These biochemical properties of A. aeolicus thermoglobin, AaTgb, may have been shared by the ancestral hemoglobin, thus suggesting possible primordial functions and providing a starting point for consequent evolution of the hemoglobin family.

Amino Acid Sequence↗

PubMed2005

Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis.

HemAT from Bacillus subtilis is a new type of heme protein responsible for sensing oxygen. The structural and functional properties of the full-length HemAT protein, the sensor domain (1-178), and Tyr-70 mutants have been characterized. Kinetic and equilibrium measurements reveal that both full-length HemAT and the sensor domain show two distinct O(2) binding components. The high-affinity component has a K(dissociation) approximately 1-2 microM and a normal O(2) dissociation rate constant, k(O2) = 50-80 s(-1). The low-affinity component has a K(dissociation) approximately 50-100 microM and a large O(2) dissociation rate constant equal to approximately 2000 s(-1). The low n-value and biphasic character of the equilibrium curve indicate that O(2) binding to HemAT involves either independent binding to high- and low-affinity subunits in the dimer or negative cooperativity. Replacement of Tyr-70(B10) with Phe, Leu, or Trp in the sensor domain causes dramatic increases in k(O2) for both the high- and low-affinity components. In contrast, the rates and affinity for CO binding are little affected by loss of the Tyr-70 hydroxyl group. These results suggest highly dynamic behavior for the Tyr-70 side chain and the fraction of the "up" versus "down" conformation is strongly influenced by the nature of the iron-ligand complex. As a result of having both high- and low-affinity components, HemAT can respond to oxygen concentration gradients under both hypoxic (0-10 microM) and aerobic (50-250 microM) conditions, a property which could, in principle, be important for a robust sensing system. The unusual ligand-binding properties of HemAT suggest that asymmetry and apparent negative cooperativity play an important role in the signal transduction pathway.

Bacillus subtilis↗

PubMed2005

The origin of stark splitting in the initial photoproduct state of MbCO.

Ligand migration and binding in heme proteins have been measured by X-ray diffraction and time-resolved spectroscopy of photoproduct intermediates. In myoglobin (Mb), internal cavities serve as docking sites for carbon monoxide (CO) ligands. In these sites, the CO ligands display characteristic infrared (IR) stretching bands due to interactions with the local electrical field. In the primary docking site, a CO can reside in two opposite orientations, characterized by a doublet of infrared bands, B1 at approximately 2130 and B2 at approximately 2120 cm-1. To assign these bands to the specific orientations, we have reexamined the effects of mutating His64 and Val68 on the infrared stretching bands associated with the B1 and B2 photoproduct states. Wild-type, H64L, V68F, and H64L-V68F MbCO were selected for experimental and theoretical analyses. Fourier transform infrared (FTIR) spectroscopy and density functional theory (DFT) calculations were used to interpret the effects of the electrostatic environment on the B state bands. The imidazole side chain of His64 appears to be the primary cause of the observed Stark splitting. The high-frequency B1 band is assigned to the CO orientation in which the carbon (white atom) is directed toward the heme iron and the Nepsilon-H proton of His64. At low temperatures, CO molecules in the opposite orientational conformer, B2 with the O atom (red) toward His64, first rotate by 180 degrees into the more stable B1 state and then rebind.

Carbon Monoxide↗

PubMed2004

Tyrosine B10 inhibits stabilization of bound carbon monoxide and oxygen in soybean leghemoglobin.

Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of a library of E7, E11, and B10 mutants indicate significant differences in the role of electrostatic interactions in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin, strong hydrogen bonds from several closely related conformations of the distal histidine (His(E7)) side chain preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of His(E7) is confined to a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of Tyr(B10) appears to "fix" the position of His(E7), probably by donating a hydrogen bond to the Ndelta atom of the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established by a favorable staggered geometry of the proximal histidine. The interaction between His(E7) and Tyr(B10) prevents overstabilization of bound oxygen. If hydrogen bonding from His(E7) were as strong as it is in mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport in root nodules.

Animals↗

PubMed2004

Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin.

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) approximately 1 microm(-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 degrees 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 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). 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 nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.

Animals↗

PubMed2004

No scavenging and the hypertensive effect of hemoglobin-based blood substitutes.

The major pathway for nitric oxide scavenging in red cells involves the direct reaction of the gas with HbO2 to form nitrate and the ferric form of the protein, metHb. Because both atoms of O2 are incorporated into nitrate, this process is called NO dioxygenation (NOD). The NOD reaction involves an initial, very rapid bimolecular addition of NO to bound O2 to form a transient Fe(III)-peroxynitrite complex, which can be observed spectrally at alkaline pH. This intermediate rapidly isomerizes at pH 7 (t1/2 <== 1 ms) to metHb and NO3-, which is nontoxic and readily transported out of red cells and excreted. The rate of NO consumption by intracellular HbO2 during normal blood flow is limited by diffusion up to and into the red cells and is too slow to interfere significantly with vasoregulation. In contrast, extracellular HbO2 is highly vasoconstrictive, and the resultant hypertension is a significant side effect of most hemoglobin-based blood substitutes. The major cause of this blood pressure effect seems to be the high rate of NO dioxygenation by cell-free HbO2, which can extravasate into the vessel walls and interfere directly with NO signaling between endothelial and smooth muscle cells. This interpretation is supported by a strong linear correlation between the magnitude of the blood pressure effect caused by infusion of cross-linked recombinant hemoglobin tetramers in vivo and the rate of NO dioxygenation by these proteins measured in vitro.

Animals↗

PubMed2004

Dynamics of carbon monoxide binding to CooA.

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n = 1.4, P(50) = 2.2 microm, and estimated Adair constants K(1) = 0.16 and K(2) = 1.3 microm(-1). The rates of CO binding and release are both strongly biphasic, with roughly equal amplitudes for the fast and slow phases. The association rates show a hyperbolic dependence on [CO], consistent with Pro-2 dissociation being rate-limiting. The kinetic characteristics of the transiently formed five-coordinate heme are probed via flash photolysis. These observations are integrated into a kinetic model, in which CO binding to one subunit decreases the rate of Pro-2 rebinding in the second, leading to a net increase in affinity for the second CO. The CO adduct exists in slowly interconverting "open" and "closed" forms. This interconversion probably involves the large-scale motions required to bring the DNA-binding domains into proper orientation. The combination of low CO affinity, slow CO binding, and slow conformational transitions ensures that activation of CooA only occurs at high (micromolar) and sustained (> or =1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the dioxygen-bound heme oxygenase from Corynebacterium diphtheriae: implications for heme oxygenase function.

HmuO, a heme 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 microm(-1) s(-1) and 0.22 s(-1), respectively, yielding an O(2) affinity of 21 microm(-1), which is approximately 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 O(2) binding. Resonance Raman spectra show that the Fe-O(2) vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe-O-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 degrees, the O-O bond is pointed toward the heme alpha-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O(2) and the amide nitrogen 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 of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin alpha-meso-carbon by preventing premature O-O bond cleavage.

Animals↗

PubMed2004

Picosecond time-resolved X-ray crystallography: probing protein function in real time.

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 100 ps to 3 micros. 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.

Amino Acid Substitution↗

PubMed2003

Structural dynamics of myoglobin: ligand migration and binding in valine 68 mutants.

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 O2 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.

Amino Acid Substitution↗

PubMed2003

Watching a protein as it functions with 150-ps time-resolved x-ray crystallography.

We report picosecond time-resolved x-ray diffraction from the myoglobin (Mb) mutant in which Leu29 is replaced by Phe (L29Fmutant). 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.

Amino Acid Substitution↗

PubMed2003

FeNO structure in distal pocket mutants of myoglobin based on resonance Raman spectroscopy.

FeNO vibrational frequencies were investigated for a series of myoglobin mutants using isotope-edited resonance Raman spectra of (15/14)NO adducts, which reveal the FeNO and NO stretching modes. The latter give rise to doublet bands, as a result of Fermi resonances with coincident porphyrin vibrations; these doublets were analyzed by curve-fitting to obtain the nuNO frequencies. Variations in nuNO among the mutants correlate with the reported nuCO variations for the CO adducts of the same mutants. The correlation has a slope near unity, indicating equal sensitivity of the NO and CO bonds to polar influences in the heme pocket. A few mutants deviate from the correlation, indicating that distal interactions differ for the NO and CO adducts, probably because of the differing distal residue geometries. In contrast to the strong and consistent nuFeC/nuCO correlation found for the CO adducts, nuFeN correlates only weakly with nuNO, and the slope of the correlation depends on which residue is being mutated. This variability is suggested to arise from steric interactions, which change the FeNO angle and therefore alter the Fe-NO and N-O bond orders. This effect is modeled with Density Functional Theory (DFT) and is rationalized on the basis of a valence isomer bonding model. The FeNO unit, which is naturally bent, is a more sensitive reporter of steric interactions than the FeCO unit, which is naturally linear. An important additional factor is the strength of the bond to the proximal ligand, which modulates the valence isomer equilibrium. The FeNO unit is bent more strongly in MbNO than in protein-free heme-NO complexes because of a combination of a strengthened proximal bond and distal interactions.

Animals↗