Quentin H. Gibson

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%.

Conformation, co-operativity and ligand binding in human hemoglobin.

Abstract There does not appear to be any co-operativity manifest in the four combination rate constants for the binding of nitric oxide to deoxyhemoglobin. The time-course of the observed reaction is best fitted by statistically related rates, and the numerical relation between the rate constants for the binding of the fourth molecule of carbon monoxide and the fourth molecule of nitric oxide, which can be obtained independently, also argues for a statistical relation between the nitric oxide binding rate constants. In spite of the absence of co-operativity, the normal T → R transition occurs on nitric oxide binding, as demonstrated by the release of 8-hydroxy-1,3,6-pyrene trisulfonate, and the R-state shows the normal enhancement of reactivity towards carbon monoxide as compared with the T-state (30-fold). Competition experiments between carbon monoxide and nitric oxide in which the two ligands react simultaneously with deoxyhemoglobin suggest that the switching point (T → R) occurs on the average after 2.7 molecules of nitric oxide have been bound (in 0.05 m -2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol, pH 7) and after 3 molecules of carbon monoxide (in 0.05 m -phosphate, PH 7).

Cavities and packing defects in the structural dynamics of myoglobin

Small globular proteins contain internal cavities and packing defects that reduce thermodynamic stability but seem to play a role in controlling function by defining pathways for the diffusion of the ligand/substrate to the active site. In the case of myoglobin (Mb), a prototype for structure–function relationship studies, the photosensitivity of the adduct of the reduced protein with CO, O2 and NO allows events related to the migration of the ligand through the matrix to be followed. The crystal structures of intermediate states of wild‐type (wt) and mutant Mbs show the photolysed CO to be located either in the distal heme pocket (primary docking site) or in one of two alternative cavities (secondary docking sites) corresponding to packing defects accessible to an atom of xenon. These results convey the general picture that pre‐existing internal cavities are involved in controlling the dynamics and reactivity of the reactions of Mb with O2 and other ligands, including NO.

The rates of polymerization and depolymerization of sickle cell hemoglobin

Abstract The polymerization and depolymerization of concentrated solutions of sickle cell deoxyhemoglobin were initiated by raising and lowering the temperature, and the time courses of the reactions monitored by the change in apparent turbidity. The polymerization reaction exhibits a marked lag phase followed by a rapid increase in turbidity, and is dependent on a very high power of the hemoglobin concentration, roughly the fifteenth. The depolymerization reaction exhibits no such lag, and is much less dependent on concentration. The implications of these results for polymerization models are discussed.

Nitric oxide recombination to double mutants of myoglobin: role of ligand diffusion in a fluctuating heme pocket.

Picosecond recombination of nitric oxide to the double mutants of myoglobin, His64Gly-Val68Ala and His64Gly.Val68Ile, at E7 and E11, has been studied experimentally and by computation. It is shown that distal residues have a profound effect on NO recombination. Recombination in the mutants may be explained in terms of fluctuating free volume and structure of the heme pocket. The double mutants provide insight into the effects of free volume and steric hindrance on rates of ligand rebinding following photolysis. Water molecules of the first solvation shell replace surface residues deleted by mutation and can block apparent holes in the protein structure. Thus, water molecules extend the time required for ligands to escape significantly to a nanosecond time scale, which is much longer than would be expected for an open heme pocket. Both nearly exponential (G64A68) and markedly nonexponential (native and G64I68) kinetics are observed, a result at variance with expectation from the model of Petrich et al. [Petrich, J.W., Lambry, J.C., Kuczera, K., Karplus, M., Poyart, C., & Martin, J.L. (1991) Biochemistry 30, 3975-3987], which attributes nonexponential kinetics to proximal effects.

Ligand migration and cavities within Scapharca Dimeric HbI: studies by time-resolved crystallo-graphy, Xe binding, and computational analysis.

As in many other hemoglobins, no direct route for migration of ligands between solvent and active site is evident from crystal structures of Scapharca inaequivalvis dimeric HbI. Xenon (Xe) and organic halide binding experiments, along with computational analysis presented here, reveal protein cavities as potential ligand migration routes. Time-resolved crystallographic experiments show that photodissociated carbon monoxide (CO) docks within 5 ns at the distal pocket B site and at more remote Xe4 and Xe2 cavities. CO rebinding is not affected by the presence of dichloroethane within the major Xe4 protein cavity, demonstrating that this cavity is not on the major exit pathway. The crystal lattice has a substantial influence on ligand migration, suggesting that significant conformational rearrangements may be required for ligand exit. Taken together, these results are consistent with a distal histidine gate as one important ligand entry and exit route, despite its participation in the dimeric interface.

Trematode Hemoglobins Show Exceptionally High Oxygen Affinity

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

Effects of phosphate upon co binding kinetics and NMR spectra of hemoglobin valency hybrids

Summary The effects of organic phosphates on the carbon monoxide binding and NMR spectra of the hemoglobin valency hybrids (αIIICN βII)2 and (αIIβIIICN)2, have been studied. The stripped deoxy hybrids show heterogeneity in kinetics with two spectrophotometrically distinct forms which are not in rapid equilibrium. At most wavelengths the fast component contributes about 60 to 80% of the total absorbance change and has a rate of binding similar to that of isolated chains or other rapid hemoglobins. The slow component reacts at about the same rate as native deoxyhemoglobin. Upon addition of organic phosphates, 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP), the fast components become slowly reacting. Changes in the NMR spectra by phosphate parallel the kinetic observations. The slow reacting form is barely affected by the phosphates.

Cytochrome oxidase from Pseudomonas aeruginosa. II. Reaction with copper protein

Abstract The reaction between a cytochrome oxidase and a copper protein from Pseudomonas aeruginosa has been studied by a rapid mixing technique. The data support the view that a complex is formed rapidly between the two proteins and is followed by a transfer of electrons in either direction. Reduced copper protein donates an electron to the heme c moiety of the oxidase with an apparent first-order rate constant of about 30 s−1 while the transfer in the reverse direction proceeds with a constant of about 120 s−1. The reaction between the copper protein and the heme c of the oxidase is followed by a much slower internal reaction involving electron transfer between the heme c and heme d. The kinetic data have been analyzed in terms of the thermodynamics of the interactions. This analysis indicates that the copper protein has a ΔE of about 0.038 V more positive than the heme c component, a value that compares favorably with that of 0.040 V obtained by equilibrium methods. The value of ΔE obtained by the kinetic method for the internal reaction is less precise but is reasonably close to that of 0.070 V determined by an equilibrium technique.

Distal pocket polarity in ligand binding to myoglobin: structural and functional characterization of a threonine68(E11) mutant.

Site-directed mutagenesis studies have confirmed that the distal histidine in myoglobin stabilizes bound O2 by hydrogen bonding and have suggested that it is the polar character of the imidazole side chain rather than its size that limits the rate of ligand entry into the protein. We constructed an isosteric Val68 to Thr replacement in pig myoglobin (i) to investigate whether the O2 affinity could be increased by the introduction of a second hydrogen-bonding group into the distal heme pocket and (ii) to examine the influence of polarity on the ligand binding rates more rigorously. The 1.9-A crystal structure of Thr68 aquometmyoglobin confirms that the mutant and wild-type proteins are essentially isostructural and reveals that the beta-OH group of Thr68 is in a position to form hydrogen-bonding interactions both with the coordinated water molecule and with the main chain greater than C=O of residue 64. The rate of azide binding to the ferric form of the Thr68 mutant was 60-fold lower than that for the wild-type protein, consistent with the proposed stabilization of the coordinated water molecule. However, bound O2 is destabilized in the ferrous form of the mutant protein. The observed 17-fold lowering of the O2 affinity may be a consequence of the hydrogen-bonding interaction made between the Thr68 beta-OH group and the carbonyl oxygen of residue 64. Overall association rate constants for O2, NO, and alkyl isocyanide binding to ferrous pig myoglobin were 3-10-fold lower for the mutant compared to the wild-type protein, whereas that for CO binding was little affected.(ABSTRACT TRUNCATED AT 250 WORDS)