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Q H Gibson

Publications and source records attributed to Q H Gibson.

At least 19 recordsLinked to original sources
PubMed1992

Distal pocket residues affect picosecond ligand recombination in myoglobin. An experimental and molecular dynamics study of position 29 mutants.

Time courses for intramolecular NO and O2 recombination to native and three position 29 mutants of sperm whale myoglobins were measured after laser photolysis on picosecond and nanosecond time scales. The rates for the first phase of NO recombination were 1.8, 2.5, 29, and > or = 100 ns-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, at room temperature. This order is not correlated with the overall association rate constants for NO binding which were all in the range 20-50 x 10(6) M-1 s-1 and is the opposite of that observed for the rate constants for the overall thermal dissociation of NO which were 5.0, 2.8, 0.98, and 0.21 x 10(-4) s-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, at 20 degrees C. This inverse correlation suggests that photo- and thermally dissociated ligand molecules experience similar kinetic and equilibrium barriers to rebinding. The larger side chains of Leu29 and Phe29 inhibit rapid movement of the ligand away from the iron atom facilitating geminate recombination. The smaller side chains of Val29 and Ala29 increase the space available to the ligand, decreasing the rate of geminate recombination and enhancing complete dissociation. Diffusion of NO in the distal pocket of myoglobin was simulated using a variant of the molecular dynamics program CHARMM that includes the locally enhanced sampling protocol (Elber, R., and Karplus, M. (1991) J. Am. Chem. Soc. 112, 9161-9175; Roitberg, A., and Elber, R. (1991) J. Chem. Phys. 95, 9277-9287) and the x-ray structures of Carver et al. (Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, M. L., Phillips, G. N., Jr., and Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450). Both accelerated (5,000 K) and room temperature ligands were used, and comparisons were made between simulations with a complete hydration shell surrounding the protein and those with only eight water molecules near the distal histidine. Photodissociated ligands initially move away from the heme plane, past Leu29, and toward Leu32, Phe33, Ile107, and Ile111. These theoretical results confirm that a complete description of picosecond ligand recombination must include the dynamics of ligand movement in the distal portion of the heme pocket.

Animals↗
PubMed1992

Ligand binding by the chlorocruorin from Eudistylia vancouverii.

Carbon monoxide chlorocruorin from Eudistylia vancouverii shows three distinct first-order relaxations with rates of 2.9 x 10(9) s-1, 6.5 x 10(7) s-1, and 3.2 x 10(6) s-1 (geminate reactions) and three second-order relaxations with rates of 4.7 x 10(6) M-1 s-1, 7 x 10(5) M-1 s-1, and 7 x 10(4) M-1 s-1, when studied by flash photolysis. The amplitudes of the second-order reactions depend on the extent of photolysis. This may be due to relaxation from the liganded (R) to the unliganded (T) conformation following photolysis and suggests that the combination rates contribute to cooperativity. In a stopped-flow experiment only the slowest phase with a rate of 7 x 10(4) M-1 s-1 is observed. It is assigned to binding to the T-state protein. Fragments of the native protein containing 12 and 4 hemes react like the holoprotein suggesting that the tetramer is a major cooperative unit. Oxygen binding shows three geminate relaxations with rates of 2.5 x 10(10) s-1, 3.5 x 10(7) s-1, and 4.5 x 10(6) s-1, and two second-order rates of 1.5 x 10(7) M-1 s-1 and 1 x 10(6) M-1 s-1. The amplitudes of the second-order phases do not correlate with the extent of photolysis. The results with the two ligands are consistent with an allosteric transition fast enough to compete with a rebinding rate of 500 s-1 in the R to T direction (CO rebinding) but not fast enough to compete with oxygen rebinding. There is significant heterogeneity in the R-state kinetics, but the T-state reaction is homogeneous.

Animals↗
PubMed1992

The relation of ligand binding to redox state in the hexa-heme nitrite reductase of Wolinella succinogenes.

Ligand binding reactions and the relation between redox state and ligand binding in the hexa-heme nitrite reductase of Wolinella succinogenes have been studied using laser flash photolysis. On a picosecond time scale, a rapid excursion was observed corresponding to the breaking and reforming of an iron histidine bond. With the CO derivative, a geminate reaction was observed with a rate of 3 ns-1. On a nanosecond time scale, no slower geminate reactions were observed. For the cyanide derivative, no geminate reactions were observed at either time scale. The second order reaction of CO with the enzyme had a time course consisting of two distinct components. This time course changed in form as the enzyme came to equilibrium with CO, and the slower rebinding component was replaced by a faster rebinding component. It is suggested that CO binding enhances reduction of a heme with an unusually low redox potential and opens the structure of the active site to allow a faster second order reaction of CO. The proportion of the geminate CO reaction was unchanged, consistent with changes relatively remote from the ligand binding site. The second order reactions of cyanide also showed that redox effects influence its rebinding reaction. Adding cyanide to the CO complex of nitrite reductase showed that the two ligands have distinct heme binding sites.

Binding Sites↗
PubMed1991

Studies of the primary oxygen intermediate in the reaction of fully reduced cytochrome oxidase.

The formation and disappearance of a photosensitive species during the reaction of reduced cytochrome c oxidase (putatively a3II.O2), EC 1.9.3.1, has been followed by (a) mixing a3II.CO with O2 in a stopped flow apparatus; (b) initiating the oxygen-oxidase reaction by removing CO with a laser flash; (c) probing the reaction mixture for photosensitivity with a second laser flash. Photosensitivity appears in the reaction mixture after the first laser flash, reaches a maximum after 50-60 microseconds ([O2] greater than 100 microM), and disappears in a further 50-100 microseconds. The kinetics can be represented by the scheme [formula: see text]. In species B, O2 is associated with the protein, possibly CuB, but not with the heme. Species C is the photosensitive a3II.O2 complex, and in D, a3 iron has been oxidized. The formation of species C is responsible for the rapid phase of absorbance change in the oxidase-oxygen reaction. The rate of reaction with oxygen approaches the limit of 35,000 s-1 at high oxygen. Nitric oxide, however, reacts with FeII oxidase with a rate of 1 x 10(8) M-1 s-1, which is accurately maintained up to an observed rate of 10(5) s-1. In flash photolysis experiments, approximately half of the photodissociated nitric oxidase recombines in a biphasic geminate reaction with rates of 1 x 10(8) s-1 and 1 x 10(7) s-1.

Animals↗
PubMed1991

Ligand binding in a hierarchy of globin complexes. The hexagonal bilayer hemoglobin of Lumbricus terrestris and its heme-containing subunits.

The kinetics of CO and NO recombination with the giant approximately 3600-kDa hexagonal bilayer hemoglobin of Lumbricus terrestris and its subunits, the approximately 200-kDa dodecamer of globin chains (3 x chains (I + II + III + IV] (see preceding paper (Vinogradov S.N., Sharma, P.K., Qabor, A.N., Wall, J.S., Westrick, J.A., Simmons, J.H., and Gill, S.J. (1991) J. Biol. Chem. 266, 13091-13096], the 50-kDa disulfide-bonded trimer (chains II-IV), the monomer (chain I), and the approximately 30-kDa linker (chains VA, VB, and VI), were measured following photolysis over time scales ranging from picosecond to millisecond. CO recombination at 436 nm subsequent to excitation (9 ns) at 532 nm showed three phases covering a 100-fold range for the Hb, dodecamer, trimer, and linker protein. The proportion of the fast phase was 0.1-0.2 for the trimer, dodecamer, and Hb. The relative rates and amplitudes of the phases were not affected by changes in CO concentration or excitation intensity. The monomer showed a single phase with a rate of 2 x 10(6) M-1 s-1. The second-order reaction with NO showed two rates. The faster rate was 90 x 10(6) M-1 s-1 and accounted for approximately 0.7 of the reaction for all species except the monomer, where it accounted for the full reaction. The slower rate was 15 x 10(6) M-1 s-1 for all species except the monomer.

Animals↗
PubMed1991

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)

Base Sequence↗
PubMed1991

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin.

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques.

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Ligand binding to a hemoprotein lacking the distal histidine. The myoglobin from aplysia limacina (Val(E7)).

The time course of ligand recombination to the myoglobin from Aplysia limacina, which has Val(E7), was measured following photolysis by flashes of 35 ps to 300 ns with a time resolution of 10 ps or 1 ns. CO shows only biomolecular recombination. O2 has a small geminate reaction with a half-time of tens of picoseconds, but no nanosecond geminate reaction. NO has two picosecond relaxations with half-times of 70 ps (15%) and 1 ns (80%) and one nanosecond relaxation with a half-time of 4.6 ns. The biomolecular rates for O2 and NO are the same: 2 x 10(7) M-1 s-1. Methyl and ethyl isonitriles have a geminate reaction with a half-time of 35 ps. Ethyl isonitrile has, in addition, a nanosecond relaxation (25%) with a half-time of 100 ns. t-Butyl isonitrile has four geminate relaxations (10 ps, 35 ps, 1 ns, and 1 microseconds). Analysis of the results suggests much easier movement of ligand between the heme pocket and the exterior than in sperm whale myoglobin (His(E7]. The reactivity of the heme is little different, placing the effect of the differences from sperm whale myoglobin on the distal side of the heme.

Animals↗
PubMed1990

Kinetics of ligand binding to haemoproteins.

A mass of experimental data has been accumulated in the 65 years since Hartridge and Roughton made the first measurement of the rapid reaction of haemoglobin with O2 in solution on a millisecond time scale, at first by flow-mixing methods, and, for 30 years or so, by flash photolysis. Technical advances, particularly in lasers, have allowed increasingly rapid reactions to be followed and the fastest reactions now observed have half-times conveniently measured in pico-seconds. The measurements were used at first to discuss the physiology of gas transport and to describe co-operativity in haemoglobin. More recently, the process of ligand binding has been dissected into intramolecular and intermolecular components. Relating the various rates to the abundance of structural information on crystals is so difficult that the work has barely begun, but the combination of kinetic measurements with genetic engineering and crystallography has promise, as well as problems, for the future.

Animals↗
PubMed1989

Ligand binding to the dimeric hemoglobin from Scapharca inaequivalvis, a hemoglobin with a novel mechanism for cooperativity.

The homodimeric hemoglobin from Scapharca inaequivalvis has an unusual spatial arrangement of the subunits (Royer, W.E., Jr., Love, W.E., and Fenderson, F.F. (1985) Nature 316, 277-280). The time course of oxygen and nitric oxide rebinding to this protein following flash photolysis has been measured on a nanosecond time scale. A large amplitude is observed with a half-time of 20 ns (NO). With oxygen the half-time decreases from 70 ns at low fractional photolysis to 30 ns at large breakdown. The second order rate of NO binding is 1.6 x 10(7)/MS, and is the same as that for oxygen. Analysis of the geminate data suggests that oxygen and nitric oxide react more rapidly with the heme than in myoglobin, but also escape much more rapidly from its vicinity.

Animals↗
PubMed1989

Ligand binding to cytochrome c peroxidase from Pseudomonas aeruginosa.

The high potential heme site of Pseudomonas cytochrome c peroxidase has His and Met as ligands. On reduction, the Fe-met bond becomes photosensitive. Following photolysis, the bond reforms with a half-time of 35 ps. The low potential heme peroxidatic site of the fully reduced enzyme has been shown to bind to a range of ligands. The compounds with carbon monoxide, methyl, ethyl, n-butyl, and t-butyl isonitriles have been investigated by laser flash photolysis. All are photosensitive and show different degrees of geminate recombination of ligand in the picosecond and nanosecond time ranges. Carbon monoxide shows the least effect. The three straight-chain isonitriles show about 50% geminate recombination with half-times of the order of 10 ns. t-Butyl isonitrile shows more and faster recombination. These results imply considerable freedom of movement within the active site for the smaller ligands.

Carbon Monoxide↗
PubMed1989

Flash photolysis of the serum albumin-heme-CO complex.

Protoheme-CO in aqueous solution does not exhibit a geminate ligand recombination reaction. Addition of a protein, either globin or serum albumin, to which heme binds strongly, leads to an observable geminate reaction in aqueous solution. The bimolecular kinetic data for the albumin-heme-CO complex show two stable components, one heme-like in rate and difference spectrum, and one hemoglobin-like. The geminate reaction correlates spectrally with the hemoglobin-like component.

Binding Sites↗
PubMed1989

The kinetics of ligand binding to plant hemoglobins. Structural implications.

The rates of reaction of oxygen, carbon monoxide, and nitric oxide with 14 plant hemoglobins have been determined by relaxation and stopped-flow methods. The combination rates for oxygen lie between 0.12 and 0.26 x 10(9)/M.s, for carbon monoxide between 0.01 and 0.07 x 10(9)/M.s, and for nitric oxide between 0.12 and 0.25 x 10(9)/M.s. The dissociation velocities for oxygen range from 5 to 25/s, and for CO from 0.005 to 0.011 s. The oxygen dissociation constants range only from 36 to 78 nM. Nanosecond relaxation experiments show large differences between the proteins. Five have known primary structures which correlate closely with the nanosecond relaxations and less immediately with the millisecond reactions. The relevant amino acid substitutions are concentrated in the C-E interhelical region.

Amino Acid Sequence↗
PubMed1988

A comparison of the geminate recombination kinetics of several monomeric heme proteins.

The geminate rate constants for CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide rebinding to soybean leghemoglobin and monomeric component II of Glycera dibranchiata hemoglobin were measured at pH 7, 20 degrees C using a dye laser with a 30-ns square-wave pulse. The results were compared to the corresponding parameters for sperm whale myoglobin and the isolated alpha and beta subunits of human hemoglobin (Olson, J.S., Rohlfs, R.J., and Gibson, Q.H. (1987) J. Biol. Chem., 262, 12930-12938). The rate-limiting step for O2, NO, and isonitrile binding to all five proteins is ligand migration up to the initial geminate state, and the rate of this process determines the overall bimolecular association rate constant for these ligands. In contrast, iron-ligand bond formation limits the overall bimolecular rate for CO binding. The distal pockets in leghemoglobin and in Glycera HbII are approximately 10 times more accessible kinetically to diatomic ligands than that in sperm whale myoglobin. This difference accounts for the much larger association rate constants (1-2 x 10(8) M-1 s-1) that are observed for O2 and NO binding to leghemoglobin and Glycera HbII. The rates of isonitrile migration through leghemoglobin are also very large and indicate a very fluid or open distal structure near the sixth coordination position. In contrast, there is a marked decrease in the rate of migration up to and away from the sixth coordination position in Glycera HbII with increasing ligand size. These results were also used to interpret previously published rate constants and quantum yields for the high (R) and low (T) affinity states of human hemoglobin. In contrast to the differences between the monomeric proteins, the differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.

Algorithms↗
PubMed1987

Geminate ligand recombination as a probe of the R, T equilibrium in hemoglobin.

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.

Binding Sites↗
PubMed1987

Ligand recombination to the alpha and beta subunits of human hemoglobin.

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.

Carbon Monoxide↗