The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO, the equilibrium form of deoxyHbA is encapsulated and only after the sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pulsed laser sources and that one of the tested encapsulation protocols is far superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.

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Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O(2) and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 microM(-1) s(-1)) and dissociation (<0.006 s(-1)) of O(2) are among the slowest known, precluding transport or diffusion of O(2) as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants for O(2) and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the chi((2,1)) dihedral angle for the indole ring increases progressively from approximately 93 degrees to at least 100 degrees in going sequentially from the deoxy to CO to O(2) derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O(2) dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.

We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. The method allows for direct comparison of functional and spectroscopic properties of equilibrium and non-equilibrium populations under the same temperature and viscosity conditions. The results implicate tertiary structure changes that include the proximal heme environment in the mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of (Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the degree of separation between the F helix and the heme is assigned as the conformational coordinate that modulates both this frequency and the innermost barrier controlling CO rebinding. A comparison with the behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are qualitatively similar to the tertiary changes that occur within both the R and T quaternary states. Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix. Myoglobin (Mb)1 continues to be used as a model protein system for investigations into how structure, dynamics, and reactivity interconnect to give rise to functionality. Recently there has been a renewed interest in myoglobin based on several new developments. From a functional point of view there are indications and suggestions that, apart from its physiological importance in facilitating oxygen diffusion, myoglobin has functions arising from its ability to participate in catalytic multisubstrate reactions involving such molecules as NO, O 2 , and H 2 O 2 (1-3). In addition the presence of apolar intra-protein substrate docking sites (the so-called Xe cavities) (4, 5) have recently been linked both to these newly proposed functions and to the kinetic patterns associated with geminate and bimolecular ligand binding (3, 6 -13).These new developments highlight fundamental biophysical issues that have not been fully addressed despite the extensive research effort that has been directed toward myoglobin. The issue that we address in this work relates to ligand reactivity as a function of conformation. Several studies show that myoglobin can adopt different tertiary conformations. In particular there is evidence that the equilibrium distribution of tertiary conformations is different for the deoxy (ferrous five coordinate) and CO derivatives of Mb. X-ray crystallographic studies show that there is a small clam shell-like rotation of the E and F helices in going from deoxy to COMb (14) as well as adjustments in the positioning of residues in the distal heme pocket (8 -10, 15, 16) that may or may not be related to the clam shell motion. Recent time-resolved x-ray crystallographic stud...

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.

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 cryogeniclike 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 3 65°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 3 A, C 3 A, and D 3 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 3 A and C 3 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.Proteins are inherently complex materials, and even their simplest reactions exhibit layers of complexity that were not anticipated a few decades ago (1-3). A case in point is carbon monoxide (CO) binding to the heme iron atom in myoglobin (Mb) 3 (4). Much of the work on Mb has been directed toward understanding the biophysical principles associated with both bimolecular and internal ligand rebinding after photolysis of the Fe-CO bond. Key issues include the following: (i) the roles of distal and proximal heme pocket amino acids; (ii) the roles of internal water molecules near the active site; (iii) the roles of local and global conformational relaxations that are modulated by solvent; and (iv) the roles of pre-existing internal cavities associated with xenon binding. Time-resolved spectroscopic and x-ray crystallographic studies have demonstrated that dissociated ligands can access internal cavities that arise from packing defects in the globin tertiary structure (5-26). A remaining challenge is to establish quantitatively how all these factors contribute to the multiple kinetic phases associated with internal ligand binding at cryogenic temperatures and high viscosity and to those observed at ambient temperatures and physiologically relevant viscosities (21, 27-37).Much of the cryogenic work and the time-resolved x-ray crystallograp...

Oxygen binding curves of sol-gel-encapsulated deoxy human adult hemoglobin (HbA) have previously revealed two distinct noncooperative populations with oxygen binding affinities approximately 1000 and 100 times lower than that of the high-affinity R state. The two populations which have been termed the low-affinity (LA) and high-affinity (HA) T states can be selectively stabilized using two different encapsulation protocols for deoxy-HbA. The present study seeks to understand the factors giving rise to these different affinity states. Visible and UV resonance Raman spectroscopies are used to characterize the conformational properties of both the deoxy and deoxy-turned-carbonmonoxy (CO) derivatives of HbA derived from the two encapsulation protocols. The geminate and bimolecular recombination of CO to the photodissociated CO derivatives is used to characterize the functional properties of the slowly evolving encapsulated populations. The results show that the initial deoxy-HbA populations are conformationally indistinguishable with respect to encapsulation protocol. The addition of CO to sol-gel-encapsulated deoxy-HbA triggers a detectable progression of conformational and functional changes. Visible resonance Raman spectra of the CO photoproduct reveal a progression of changes of the iron-proximal histidine stretching frequencies: 215, 222, 227, and 230 cm(-1). The low and high values correspond to the initial deoxy T state and liganded R (R(2)) state species, respectively. The 222 and 227 cm(-1) species are generated using encapsulation protocols that give rise to what are termed the LA and HA T states, respectively. The UV resonance Raman spectra of these and related species indicate that the progression from deoxy T to LA to HA is associated with a progressive loosening of T state constraints within the hinge and switch regions of the alpha(1)beta(2) interface. The time scale for the progression is determined by a balance between the ligation-initiated evolution toward high-affinity conformations and factors such as allosteric effectors, gel matrix, and added glycerol that slow ligand-binding-induced relaxation. Thus, it appears that the encapsulation protocol-dependent rate of ligand-binding-induced relaxation determines the functional properties of the initially encapsulated deoxy-HbA population.

The reactive sulfhydryl on Cys beta93 in human adult hemoglobin (HbA) has been the focus of much attention. It has purported functional roles such as a transporter of nitric oxide and a detoxifier of super oxide. In addition, it has a proposed role in the allosteric mechanism. The present study addresses the functional and conformational consequences of modifying the beta93 sulfhydryl using either maleimide or disulfide-based reactions. The geminate and bimolecular recombination of CO derivatives of several different beta93-modified Hbs in both solution and sol-gel matrixes provide a window into functional modifications associated with both the R and T states of these proteins. Nanosecond time-resolved visible resonance Raman spectroscopy is used to probe conformational consequences associated with the proximal heme environment. The results show functional and conformational consequences that depend on the specific chemistry used to modify beta93. Maleimide-based modification show the most significant alterations of R-state properties including a consistent pattern of a reduced geminate yield and a loss of the favorable heme-proximal histidine interaction normally seen for liganded R-state HbA. A mechanism based on a displacement of the side chain of Tyr beta145 is explored as a basis for this effect as well as other situations where there is loss of the quaternary enhancement effect. The quaternary enhancement effect refers to the enhancement of ligand binding properties of the alphabeta dimers when they are associated into the R-state tetramer.