John S. Olson

Mapping the Pathways for O2 Entry Into and Exit from Myoglobin

The effects of mutagenesis on geminate and bimolecular O2 rebinding to 90 mutants at 27 different positions were used to map pathways for ligand movement into and out of sperm whale myoglobin. By analogy to a baseball glove, the protein “catches” and then “holds” incoming ligand molecules long enough to allow bond formation with the iron atom. Opening of the glove occurs by outward movements of the distal histidine (His64), and the ligands are trapped in the interior “webbing” of the distal pocket, in the space surrounded by Ile28, Leu29, Leu32, Val68, and Ile107. The size of this pocket is a major determinant of the rate of ligand entry into the protein. Immediately after photo- or thermal dissociation, O2 moves away from the iron into this interior pocket. The majority of the dissociated ligands return to the active site and either rebind to the iron atom or escape through the His64 gate. A fraction of the ligands migrate further away from the heme group into cavities that have been defined as Xe binding sites 4 and 1; however, most of these ligands also return to the distal pocket, and net escape through the interior of wild-type myoglobin is <20–25%.

Kinetic Pathways and Barriers for Ligand Binding to Myoglobin

Myoglobin is a small globular heme protein that increases the aerobic capacity of striated vertebrate muscle cells by taking up oxygen from blood during rest and delivering O2 to mitochondria during muscle contraction when blood flow through capillaries is restricted. The ferrous form of myoglobin can also react with CO and NO, which are produced in vivo as second messengers for regulating various physiological functions including blood pressure, platelet aggregation, and neurotransmission. Its tertiary structure consists of eight tightly packed helices, and the resulting “myoglobin fold” is very similar to that found for the a and b subunits of hemoglobin. Since Gibson’s minireview in 1989, a number of exciting new studies have led to detailed molecular mechanisms of myoglobin function. The rapid progress made in the past 7 years is primarily the result of ultrafast kinetic measurements, mutagenesis experiments, and theoretical molecular dynamics simulations. Significant contributions have been made individually by these approaches, but more progress has occurred when these endeavors have been combined (e.g. Gibson et al., 1992; Braunstein et al., 1993; Schlichting et al., 1994; Teng et al., 1994; Huang and Boxer, 1994; Petrich et al., 1994; Carlson et al., 1994; Quillin et al. 1995). This review focuses on NO, O2, and CO binding to myoglobin mutants under physiological conditions. The objectives are to summarize successes in correlating theoretical, structural, and kinetic results and to identify the major remaining questions in ligand binding dynamics.

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 KCO 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 KNO 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 KO2, KCO, and KNO 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.

The role of the distal histidine in myoglobin and haemoglobin

The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins1–8. A hydrogen bond between the Nɛ proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron1–8. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides2–8. To test these ideas, engineered mutants of sperm whale myoglobin9 and the α- and β-subunits of human haemoglobin8,10–12 were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the α-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to β-chains within R-state haemoglobin. In the β-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies4,5 account for the functional differences of these proteins.

Crystal structure of a nonsymbiotic plant hemoglobin.

BACKGROUND Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

Nitric-oxide Dioxygenase Activity and Function of Flavohemoglobins SENSITIVITY TO NITRIC OXIDE AND CARBON MONOXIDE INHIBITION

Widely distributed flavohemoglobins (flavoHbs) function as NO dioxygenases and confer upon cells a resistance to NO toxicity. FlavoHbs from Saccharomyces cerevisiae,Alcaligenes eutrophus, and Escherichia colishare similar spectra, O2, NO, and CO binding kinetics, and steady-state NO dioxygenation kinetics. Turnover numbers (V max) for S. cerevisiae, A. eutrophus, and E. coli flavoHbs are 112, 290, and 365 NO heme−1 s−1, respectively, at 37 °C with 200 μm O2. The K M values for NO are low and range from 0.1 to 0.25 μm.V max/K M (NO) ratios of 900–2900 μm −1 s−1 indicate an extremely efficient dioxygenation mechanism. ApproximateK M values for O2 range from 60 to 90 μm. NO inhibits the dioxygenases at NO:O2ratios of ≥1:100 and makes true K M (O2) values difficult to determine. High and roughly equal second order rate constants for O2 and NO association with the reduced flavoHbs (17–50 μm −1 s−1) and small NO dissociation rate constants suggest that NO inhibits the dioxygenase reaction by forming inactive flavoHbNO complexes. Carbon monoxide also binds reduced flavoHbs with high affinity and competitively inhibits NO dioxygenases with respect to O2(K I (CO) = ∼1 μm). These results suggest that flavoHbs and related hemoglobins evolved as NO detoxifying components of nitrogen metabolism capable of discriminating O2 from inhibitory NO and CO.

Myoglobin as a model system for designing heme protein based blood substitutes

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 MbO(2) to form NO(3)(-) and metMb. This reaction rapidly detoxifies NO and is a key physiological function of both myoglobins and hemoglobins. The mechanisms derived for O(2) binding and NO dioxygenation have been used to design safer, more efficient, and more stable heme protein-prototypes for use as O(2) 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.

Quaternary structure regulates hemin dissociation from human hemoglobin.

Rate constants for hemin dissociation from the α and β subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 °C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated α and β chains and for recombinant hemoglobin tetramers stabilized by α subunit fusion. The rate constant for hemin dissociation from β subunits in native hemoglobin increases from 1.5 h−1in tetramers at high protein concentration to 15 h−1 in dimers at low concentrations. The rate of hemin dissociation from α subunits in native hemoglobin is significantly smaller (0.3–0.6 h−1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-α subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric α and β chains are much greater, 12 and 40 h−1, respectively, at pH 7, 37 °C. Aggregation of monomers to form α1β1 dimers greatly stabilizes bound hemin in α chains, decreasing its rate of hemin loss ∼20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to β subunits. A significant reduction in the rate of hemin loss from β subunits does occur after formation of the α1β2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.

Pathway for Heme Uptake from Human Methemoglobin by the Iron-regulated Surface Determinants System of Staphylococcus aureus

The iron-regulated surface proteins IsdA, IsdB, and IsdC and transporter IsdDEF of Staphylococcus aureus are involved in heme acquisition. To establish an experimental model of heme acquisition by this system, we have investigated hemin transfer between the various couples of human methemoglobin (metHb), IsdA, IsdB, IsdC, and IsdE by spectroscopic and kinetic analyses. The efficiencies of hemin transfer from hemin-containing donors (holo-protein) to different hemin-free acceptors (apo-protein) were examined, and the rates of the transfer reactions were compared with that of indirect loss of hemin from the relevant donor to H64Y/V68F apomyoglobin. The efficiencies, spectral changes, and kinetics of the transfer reactions demonstrate that: 1) metHb directly transfers hemin to apo-IsdB, but not to apo-IsdA, apo-IsdC, and apo-IsdE; 2) holo-IsdB directly transfers hemin to apo-IsdA and apo-IsdC, but not to apo-IsdE; 3) apo-IsdE directly acquires hemin from holo-IsdC, but not from holo-IsdB and holo-IsdA; and 4) IsdB and IsdC enhance hemin transfer from metHb to apo-IsdC and from holo-IsdB to apo-IsdE, respectively. Taken together with our recent finding that holo-IsdA directly transfers its hemin to apo-IsdC, these results provide direct experimental evidence for a model in which IsdB acquires hemin from metHb and transfers it directly or through IsdA to IsdC. Hemin is then relayed to IsdE, the lipoprotein component of the IsdDEF transporter.

Review: Correlations between oxygen affinity and sequence classifications of plant hemoglobins

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full‐length globins with the classical eight helix Mb‐like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O2 binding (∼250 μM−1 s−1), moderately high rates of O2 dissociation (∼5–15 s−1), and high oxygen affinity (Kd or P50 ≈ 50 nM). These properties both facilitate O2 diffusion to respiring N2 fixing bacteria and reduce O2 tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (KHisE7 ≈ 2), moderate rates of O2 binding (∼25 μM−1 s−1), very small rates of O2 dissociation (∼0.16 s−1), and remarkably high O2 affinities (P50 ≈ 2 nM), suggesting a function involving O2 and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (KHisE7 ≈ 100), low rates of O2 binding (∼1 μM−1 s−1), moderately low O2 dissociation rate constants (∼1 s−1), and moderate, Mb‐like O2 affinities (P50 ≈ 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.