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J S Olson

Publications and source records attributed to J S Olson.

At least 55 records · Page 3Linked to original sources
PubMed1995

The D-helix in myoglobin and in the beta subunit of hemoglobin is required for the retention of heme.

All globins consist of eight helices and interconnecting loops except alpha hemoglobin subunits which lack the D-helix due to deletion of five consecutive residues. Previous site-directed mutagenesis work suggested that this deletion is a neutral modification in hemoglobin with respect to equilibrium O2 binding [Komiyama, N. H., Shih, T.-B., Looker, D., Tame, J., & Nagai, K. (1991) Nature 352, 349-351]. To examine the role of the D-helix in myoglobin, we have measured the O2 and CO binding and hemin dissociation properties of recombinant sperm whale myoglobin mutants in which residues 52-56 have been deleted, Mb(-D), replaced by five alanines, Mb(Ala52-56), and substituted with four alanines and a methionine, Mb(Ala52-55Met56). Crystal structures of aquometMb(-D) and aquometMb(Ala52-55Met56) were determined to 2.0 A resolution and show that the conformation of the distal pocket is little affected by removal of the D-helix or mutations in this region. As a result, these mutations have little effect on O2 and CO binding. Diffuse electron density is observed in the region between the C- and E-helices of Mb(-D), indicating a highly mobile or heterogeneous conformation in this portion of the tertiary structure. This flexibility provides an explanation for the 50-fold higher rate of hemin loss from Mb(-D) as compared to that from wild-type myoglobin. Hemin loss from Mb(Ala52-56) is also rapid. In contrast, Mb(Ala52-55Met56) shows a well-defined D-helix and has a rate of hemin loss identical to that of wild-type holoprotein [corrected].(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Structural and functional effects of apolar mutations of the distal valine in myoglobin.

High-resolution structures of the aquomet, deoxy, and CO forms of Ala68, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild-type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O2, CO, and NO binding. Molecular dynamics simulations based on crystal structures have provided information about the time-dependent behavior of photolyzed ligands for comparison with picosecond geminate recombination studies. The Val68-->Ala mutation has little effect on the structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N epsilon atom of the distal histidine restricts ligand binding and appears to be more important in regulating the function of myoglobin than direct steric interactions between the ligand and the C gamma atoms of the native valine side-chain. This distal pocket water molecule is displaced by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate about its C alpha-C beta and C beta-C gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picosecond NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side-chain is restricted due to branching at the beta carbon, and as a result, the delta methyl group is located close to the iron atom in both the deoxy and liganded structures. The favorable effect of displacing the distal pocket water molecule is offset by direct steric hindrance between the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three ligands and inhibition of geminate recombination which is reproduced in the molecular dynamics simulations. In Phe68 myoglobin, the benzyl side-chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobins, a well-defined water molecule is found hydrogen bonded to the distal histidine in Phe68 deoxymyoglobin. This water molecule, in combination with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. (ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Phe-46(CD4) orients the distal histidine for hydrogen bonding to bound ligands in sperm whale myoglobin.

The role of Phe-46(CD4) in modulating the functional properties of sperm whale myoglobin was investigated by replacing this residue with Leu, Ile, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid almost makes direct contact with the distal histidine and has been postulated to affect ligand binding. The overall association rate constants for CO, O2, and NO binding were little affected by decreasing the size of residue 46 step-wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O2, and NO dissociation increased 4-, 10-, and 25-fold, respectively, for the same series of mutants, causing large decreases in the affinity of myoglobin for all three diatomic gases. The rates of autooxidation at 37 degrees C, pH 7.0 increased dramatically from approximately 0.1-0.3 h-1 for wild-type, Tyr-46, and Trp-46 myoglobins to 1.5, 5.2, 4.9, and 5.0 h-1 for the Leu-46, Ile-46, Val-46 and Ala-46 mutants, respectively. Rates of NO and O2 geminate recombination were measured using 35 ps and 9 ns laser excitation pulses. Decreasing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolution structures of Leu-46 and Val-46 metmyoglobins, Val-46 CO-myoglobin, and Val-46 deoxymyoglobin were determined by X-ray crystallography. When Phe-46 is replaced by Val, the loss of internal packing volume is compensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E-helix toward the mutation site, (3) greater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal histidine (His-64). In wild-type myoglobin, the van der Waals contact between C zeta of Phe-46 and C beta of His-64 appears to restrict rotation of the imidazole side chain. Insertion of Val at position 46 relieves this steric restriction, allowing the imidazole side chain to rotate about the C alpha - C beta bond toward the surface of the globin and about the C beta - C gamma bond toward the space previously occupied by the native Phe-46 side chain. This movement disrupts hydrogen bonding with bound ligands, causing significant decreases in affinity, and opens the distal pocket to solvent water molecules, causing marked increases in the rate of autooxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Identification of the iron-carbonyl stretch in distal histidine mutants of carbonmonoxymyoglobin.

Soret-excitation resonance Raman (RR) spectra are reported for six distal histidine mutants of carbonmonoxymyoglobin including H64A, H64V, H64L, H64I, H64W, and H64W/L29F. Based on 13CO isotope shifts, the iron-carbonyl stretching vibrations are unambiguously identified. The correct assignment of these modes eliminates the differences in the conformational substate occupations predicted by the RR versus IR data.

Binding Sites↗
PubMed1994

Stability of myoglobin: a model for the folding of heme proteins.

Factors governing the stability of sperm whale, pig, and human metmyoglobin were examined by (1) measuring guanidinium chloride induced unfolding of apoglobins containing 22 replacements at positions 29(B10), 43(CD1), 64(E7), 68(E11), and 107(G8), (2) determining the rates of hemin loss from the recombinant holoproteins, and (3) estimating constitutive expression levels of the corresponding genes in Escherichia coli TB-1 cells. The denaturant titrations were analyzed in terms of a two-step unfolding reaction, N(native apoprotein)-->I(intermediate)-->U(unfolded), in which the intermediate is visualized by an increase in tryptophan fluorescence emission. Two key conclusions were reached. First, high rates of hemin loss are not necessarily correlated with unstable globin structures and vice versa. In general, both rates of hemin loss and the equilibrium constants for apoprotein unfolding must be determined in order to understand the overall stability of heme proteins and to predict the efficiency of their expression. Second, polar residues in the distal pocket cause marked decreases in the overall stability of apomyoglobin. Removal of hemin from V68N and L29N sperm whale myoglobins produces the molten globular I state at pH 7, 25 degrees C, without addition of denaturant. In contrast, the H64L and H64F mutations produce apoproteins which are 10-30 times more stable than wild-type apoglobin. The latter results show that protein stability is sacrificed in order to have the distal histidine (H64) present to increase O2 affinity and inhibit autooxidation.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

L-arginine and calmodulin regulation of the heme iron reactivity in neuronal nitric oxide synthase.

Neuronal nitric oxide synthase (NOS) is a calmodulin-dependent, flavin-containing hemoprotein that forms NO from L-arginine, NADPH, and molecular oxygen. Calmodulin binding to NOS triggers reduction of its heme groups (Abu-Soud, H., and Stuehr, D.J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10762), leading to NADPH oxidation and NO synthesis. We have examined how L-arginine and calmodulin control the ligand binding and electron acceptor properties of the NOS heme iron. In the absence of bound calmodulin, ferric NOS exhibited a Kd of 0.6 microM for L-arginine, as determined by the substrate-dependent shift in heme spin equilibrium toward a high spin state. L-Arginine binding reduced the affinity of the ferric NOS heme for cyanide by 8-fold. Carbon monoxide binding to substrate-free ferrous NOS occurred at a rate of 2 x 10(5) M-1 S-1; this rate was decreased 12-fold when L-arginine was bound. In contrast, bound calmodulin did not significantly affect cyanide or carbon monoxide binding to the NOS heme, nor did it alter NOS binding affinity for L-arginine. Anaerobic titration of a calmodulin-bound, L-arginine-free NOS with NADPH led to incomplete reduction of the heme iron; full reduction was achieved only in the presence of added L-arginine. Thus, our data suggest that L-arginine controls NOS heme iron reactivity in at least two ways: 1) it slows ligand interactions by binding in the distal pocket very near the heme and 2) it also appears to increase the reduction potential of the iron. In contrast, bound calmodulin does not alter the NOS affinity for L-arginine or heme ligands and may function solely as a switch that enables electrons to pass from the flavin domain onto the heme iron.

Amino Acid Oxidoreductases↗
PubMed1994

Structural determinants of the stretching frequency of CO bound to myoglobin.

In order to assess the relative importance of polar versus steric interactions, infrared spectra and overall CO binding properties were measured at room temperature for 41 different recombinant myoglobins containing mutations at His64(E7), Val68(E11), Phe43(CD1), Arg45(CD3), Phe46(CD4), and Leu29(B10). The results were compared to the crystal structures of wild-type, Phe29, Val46, Ala68, Phe68, Gln64, Leu64, and Gly64 sperm whale CO-myoglobin and that of Thr68 pig CO-myoglobin. As observed in several previous studies, replacement of the distal histidine (His64) with aliphatic amino acids results in the appearance of a single IR band in the 1960-1970-cm-1 region and in large increases in CO affinity (KCO). More complex behavior is observed for Gly, Ala, Gln, Met, and Trp substitutions at position 64, but in each case there is a net increase in the intensity of this high-frequency component. Replacement of Val68 with Ala, Leu, Ile, and Phe produces little effect on the IR spectrum, whereas these mutations cause 20-fold changes in KCO, presumably due to steric effects. Replacement of Val68 with Thr decreases KCO 4-5-fold, whereas the position of the major IR band increases from 1945 to 1961 cm-1. Replacement of Val68 with Asn also causes a large decrease in KCO, but in this case, the peak position of the major IR band decreases from 1945 to 1916 cm-1. Nine replacements were made in the CD corner at positions 43, 45, and 46. All of the resultant mutants show increased stretching frequencies that can be correlated with movement of the imidazole side chain of His64 away from the bound ligand. All five substitutions at position 29 cause changes in the IR spectra. The Leu29-->Phe mutation had the largest effect, producing a single band centered at 1932 cm-1. Together these data demonstrate that there is little direct correlation between affinity, vCO, and Fe-C-O geometry. The major factor governing vCO appears to be the electrostatic potential surrounding the bound ligand and not steric hindrance. The presence of positive charges from proton donors, such as N epsilon of His64 and N delta of Asn68, cause a decrease in the bond order and stretching frequency of bound CO. In contrast, the negative portion of the Thr68 dipole points directly toward the bound ligand and increases the C-O bond order and stretching frequency. Movement of His64 away from the bound ligand or replacement of this residue with aliphatic amino acids prevents hydrogen-bonding interactions, causing vCO to increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

His64(E7)-->Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation.

To develop an assay for hemin dissociation, His64(E7) was replaced by Tyr in sperm whale myoglobin producing a holoprotein with a distinct green color due to an intense absorption band at 600 nm. Val68(E11) was replaced by Phe in the same protein to increase its stability. When excess Tyr64-Val68 apoglobin is mixed with either metmyoglobin or methemoglobin, the solution turns from brown to green, and the absorbance changes can be used to measure complete time courses for hemin dissociation from either holoprotein. This assay has been used to measure rates of hemin dissociation from native metmyoglobin, four myoglobin mutants (Ala64(E7), Ala68(E11), Phe68(E11), and Glu45(CD3)), native methemoglobin, valence hybrid hemoglobins, and two mutant hemoglobins ((alpha(Gly-E7)beta(native))2, and (alpha(native)beta(Gly-E7))2). Two kinetic phases were observed for hemin dissociation from native human hemoglobin at pH 7.0 and 37 degrees C. Valence and mutant hybrid hemoglobins were used to assign the faster phase (k = 7.8 +/- 2.0 h-1) to hemin dissociation from ferric beta subunits and the slower (k = 0.6 +/- 0.15 h-1) to dissociation from alpha subunits. The corresponding rate for wild-type metmyoglobin is 0.007 +/- 0.004 h-1.

Animals↗
PubMed1994

Genetic engineering of myoglobin as a simple prototype for hemoglobin-based blood substitutes.

Site-directed mutagenesis has been used to examine the structural and functional roles of distal pocket residues in regulating O2 affinity, CO binding, rates of association and dissociation, autooxidation, and hemin loss in mammalian myoglobins and human hemoglobin. In myoglobin, His-E7 inhibits CO binding by requiring displacement of distal pocket water. In the case of O2 binding, this displacement is compensated by a strong hydrogen bond between the bound ligand and the imidazole side chain. The isopropyl side chain Val-E11 also sterically restricts CO binding. The rates of ligand binding are regulated by distal pocket water displacement, steric restrictions near the iron atom, and an outer more global protein barrier. Autooxidation occurs by two mechanisms, direct dissociation of HO2 and bimolecular reaction of external O2 with unliganded heme. Both processes are inhibited markedly by hydrogen bonding interactions with His-E7. Double mutants have been constructed to decrease oxygen affinity, but still prevent oxidation. The apoprotein of His-E7-->Tyr myoglobin has been used to extract hemin from other myoglobins and hemoglobin, causing a brown to green color change. This assay has been used to show that polar interactions between residues CD3, E7, E10, F7, and the porphyrin propionates inhibit hemin dissociation markedly.

Amino Acid Sequence↗
PubMed1993

Distal pocket polarity in ligand binding to myoglobin: deoxy and carbonmonoxy forms of a threonine68(E11) mutant investigated by X-ray crystallography and infrared spectroscopy.

The crystal structures of the deoxy and carbonmonoxy forms of a distal pocket myoglobin mutant in which valine68(E11) is replaced by threonine have been solved to 2.1- and 2.2-A resolution, respectively. This substitution has been shown previously to cause large decreases in the rate of oxygen binding and to lower the equilibrium association constants for O2 and CO. The synchrotron Laue method was used for the rapid acquisition of X-ray diffraction data to overcome problems caused by the very rapid rate of autooxidation of the mutant protein. The refined deoxy structure shows that the noncoordinated water molecule in the distal pocket is in a position to form strong hydrogen bonds with both the N epsilon-H of the distal histidine64 and O gamma of threonine68 with no other unexpected alterations in the protein structure. In the carbonmonoxy form, the bound ligand is well-defined and inclined away from the two hydrogen-bonding groups, refining to a position in which the Fe-C-O angle is 162 degrees. This value is very close to that previously observed in recombinant wild-type and position-64 (E7) mutants of sperm whale myoglobin (160-170 degrees). The similarity of the CO conformations contrasts with the 150-fold range in equilibrium binding constants (KCO) among the distal pocket myoglobin mutants and indicates that CO affinities cannot be predicted from the coordination geometry of the bound ligand. Furthermore, a comparison of the infrared stretching frequencies of CO in wild-type, valine64 and threonine68 single mutant, and valine64-threonine68 double mutant pig carbonmonoxymyoglobins shows a lack of correlation between KCO and vCO. These effects can be understood in terms of the stability of noncovalently bound water in deoxymyoglobin and electrostatic interactions between bound ligands and the distal pocket residues.

Animals↗
PubMed1993

High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin.

The highly conserved distal histidine residue (His64) of sperm whale myoglobin modulates the affinity of ligands. In an effort to fully characterize the effects of mutating residue 64, we have determined the high-resolution crystal structures of the Gly64, Val64, Leu64, Thr64 and Gln64 mutants in several liganded forms. Metmyoglobins with hydrophobic substitutions at residue 64 (Val64 and Leu64) lack a water molecule at the sixth coordination position, while those with polar amino acid residues at this position (wild-type and Gln64) retain a covalently bound water molecule. In the Thr64 mutant, the bound water position is only partially occupied. In contrast, mutating the distal histidine residue to glycine does not cause loss of the coordinated water molecule, because the hydrogen bond from the imidazole side-chain is replaced by one from a well-ordered solvent water molecule. Differences in water structure around the distal pocket are apparent also in the structures of deoxymyoglobin mutants. The water molecule that is hydrogen-bonded to the N epsilon atom of histidine 64 in wild-type deoxymyoglobin is not found in any of the position 64 mutant structures that were determined. Comparison of the carbonmonoxy structures of wild-type, Gly64, Leu64 and Gln64 myoglobins in the P6 crystal form shows that the conformation of the Fe-C-O complex is nearly linear and is independent of the identity of the amino acid residue at position 64. However, the effect of CO binding on the conformation of residue 64 is striking. Superposition of deoxy and carbonmonoxy structures reveals significant displacements of the residue 64 side-chain in the wild-type and Gln64 myoglobins, but no displacement in the Leu64 mutant. These detailed structural studies provide key insights into the mechanisms of ligand binding and discrimination in myoglobin.

Animals↗
PubMed1993

Kinetics of ligand binding to Pseudoterranova decipiens and Ascaris suum hemoglobins and to Leu-29-->Tyr sperm whale myoglobin mutant.

The kinetics of binding of O2, CO, and NO to the octameric, two-domain hemoglobins of the parasitic nematodes Pseudoterranova decipiens and Ascaris suum were determined on nanosecond and picosecond time scales using flash photolysis. The two nematode hemoglobins have very similar kinetic properties. On the picosecond time scale, they exhibit an unusual behavior in showing a geminate reaction with oxygen that is biphasic and dependent on the flash intensity. The geminate reaction with NO is also faster and more complete than for sperm whale myoglobin; however, in contrast to the O2 reaction, it is homogeneous. In addition, the oxygen dissociation rate of P. decipiens hemoglobin, 0.0035 s-1, is as low as that of A. suum hemoglobin, 0.004 s-1 (Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. Lond. B Biol. Sci. 163, 206-214). A mutant of sperm whale myoglobin suggested by sequence alignment of the nematode hemoglobins, Leu-29-->Tyr, did not have kinetic properties similar to them.

Animals↗
PubMed1993

Lysine 84 is at the subunit interface of lac repressor protein.

Mutations have been generated at the Lys84 site of the lac repressor to explore its predicted role in inducer binding and/or subunit interaction. Four single mutations, Lys84-->Ala, Lys84-->Leu, Lys84-->Arg, and Lys84-->Glu, have been generated by site-specific mutagenesis. In addition, the mutation Tyr282-->Asp, which results in a monomeric repressor, has been coupled with these four single mutants to generate the four corresponding double mutants. Unchanged inducer binding affinities in all Lys84 mutants except Lys84-->Arg suggest that Lys84 does not contribute energy to inducer binding and is not found in the inducer-binding site as previously proposed (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1984) Nature 310, 429-430). Interestingly, the double mutants with hydrophobic side chains at the Lys84 site are tetramers, while those with charged side chains remain monomers. This result agrees with the recent model of the lac repressor (Nichols, J. C., Vyas, N. K., Quiocho, F. A., and Matthews, K. S. (1993) J. Biol. Chem. 268, 17602-17612), in which Lys84 is mapped by sequence alignment to the same face of the subunit as Tyr282. More detailed inducer binding, operator binding, and immunoblotting studies show that all the mutations at Lys84 have quaternary structures that deviate from wild-type protein, providing supportive evidence for the model placing this residue on the surface of the monomer subunit. Substitution of Lys84 by Ala or Leu results in 100-200-fold decreased association and dissociation rate constants for inducer binding and biphasic character. This decrease can be rescued at least partially in the respective double mutants at elevated pH, at which wild-type repressor shows a 10-fold decrease in affinity and cooperativity in inducer binding. In all substitutions with Ala or Leu, immunoblotting patterns with monoclonal antibody, an assay sensitive to alterations in quaternary structure, are distinct from wild-type repressor. Although substitution with Arg at position 84 yields a protein with 10-fold lower inducer binding affinity, the mutant shows decreased pH dependence of inducer binding. Substitution at this site with Glu results in cooperativity at neutral pH with no change in inducer binding at elevated pH. In addition, operator binding affinity of this mutant is affected by elevated pH, a phenomenon not observed in wild-type repressor. These changes in inducer and operator binding properties appear to be related to the altered quaternary structure of these mutants at Lys84.

Chromatography, Gel↗
PubMed1993

Serine92 (F7) contributes to the control of heme reactivity and stability in myoglobin.

The effects of mutation of the conserved serine92 residue to alanine, valine, and leucine in pig myoglobin have been determined. In myoglobin crystal structures, the hydroxyl group of serine92 is within hydrogen-bonding distance of the N delta-H of histidine93, whose N epsilon coordinates the iron atom of the heme prosthetic group. The association equilibrium constants of the ferrous forms of the mutant myoglobins for O2, CO, and methyl and ethyl isocyanide are increased 1.3-13-fold relative to the wild-type protein. The rates of azide association with the mutant ferric proteins at neutral pH are decreased by factors of 2-5 consistent with an increased affinity for the iron-bound water molecule which must be displaced. The dissociation rates for azide appear to be decreased 4-10-fold, suggesting that the affinity of the mutant proteins for this ligand is also higher. Thus, the overall affinities are increased regardless of the chemical nature of the liganded species, indicating that the reactivity of the heme iron itself has been raised. Time courses for association of methyl and ethyl isocyanide at high concentrations show fast and slow phases in which the absorbance at 445 nm drops and then rises, respectively. Comparison of these traces with spectra following the reaction of isocyanide ligands with chelated proton heme in soap micelles indicates that the slow phase is associated with the breaking of the iron-proximal histidine bond and the binding of a second isocyanide species in the proximal heme pocket.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Ligand diffusion in the distal heme pocket of myoglobin. A primary determinant of geminate rebinding.

There are at least two picosecond kinetic components in the rebinding of NO to native sperm whale myoglobin. Petrich et al. (Petrich, J. W., Lambry, J.-C., Kuczera, K., Karplus, M., Poyart, C., and Martin, J.-L. (1991) Biochemistry 30, 3975-3987) attribute the slowing of the reaction to a movement of the iron atom out of the plane of the heme following ligand dissociation. In contrast, Gibson et al. (Gibson, Q. H., Regan, R., Elber, R., Olson, J. S., and Carver, T. E. (1992) J. Biol. Chem. 267, 22022-22034) have explained multiphasic geminate reactions by diffusion of NO into the distal heme pocket as determined by its detailed structure. O2 and NO rebinding to iron and cobalt derivatives of native, V68F, and V68I sperm whale myoglobin has been examined. Each iron protein shows a biphasic time course of NO rebinding reactions with widely different rates and amplitudes. Although cobalt does not move out of the plane of the porphyrin on ligand removal, the reactions of the iron and cobalt derivatives of each protein were closely similar. The time course of O2 rebinding to cobalt was also similar to that of NO rebinding to iron. These results are consistent with a primary role for the structure of the distal pocket in determining diffusion of ligands away from the metal atom and as a result the time course of picosecond ligand rebinding.

Animals↗