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...

A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different alphabeta dimer conformations within the tetramer.

The current limitations of nitric oxide (NO) delivery systems has stimulated an extraordinary interest in the development of compounds that generate NO in a controlled and sustained manner with a heavy emphasis on the treatment of cardiovascular disease states. This work describes the positive physiological response to the infusion of NO releasing nanoparticles prepared using a new platform based on hydrogel/glass hybrid nanoparticles. When exposed to moisture, these nanoparticles slowly release therapeutic levels of NO, previously generated through thermal reduction of nitrite to NO trapped within the dry particles. The controlled and sustained release of NO observed from these nanoparticles (NO-np) is regulated by its hydration over extended periods of time. In a dose-dependent manner, circulating NO-np both decreased mean arterial blood pressure and increased exhaled concentrations of NO over a period of several hours. Circulating NO-np induced vasodilatation and increased microvascular perfusion during their several hour circulation lifetime. Control nanoparticles (Control-np; without nitrite) did not induce changes in arterial pressure, although a decrease in the number of capillaries perfused and increase in leukocyte rolling and immobilization in the microcirculation was observed. The NO released by the NO-np prevents the inflammatory response observed after infusion of Control-np. These data suggest that NO release from NO-np is advantageous relative to other NO releasing compounds, because it does not depend on chemical decomposition or enzymatic catalysis; it is only determined by the rate of hydration. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent and as a research tool to increase our understanding of NO signaling mechanisms within the vasculature.

Ligand recombination studies play a central role both for characterizing different hemeproteins and there conformational states but also for probing fundamental biophysical processes. Consequently, there is great importance to providing a foundation from which one can understand the physical processes that give rise to and modulate the large range of kinetic patterns associated with ligand recombination in myoglobins and hemoglobins. In this work, an overview of cryogenic and solution phase recombination phenomena for COMb is first reviewed and then a new paradigm is presented for analyzing the temperature and viscosity dependent features of kinetic traces in terms of multiple phases that reflect which tier(s) of solvent-slaved protein dynamics is(are) operative on the photoproduct population during the time course of the measurement. This approach allows for facile inclusion of both ligand diffusion among accessible cavities and conformational relaxation effects. The concepts are illustrated using kinetic traces and MEM populations derived from the CO recombination process for wild type and mutant myoglobins either in sol-gel matrices bathed in glycerol or in trehalose derived glassy matrices. Keywords myoglobin; carbonmonoxide; sol-gel; trehalose; geminate recombination; Xe cavities Ligand recombinationStudies utilizing ligand recombination subsequent to photodissociation continue to provide basic insight into the functional properties of the many varieties of hemoglobin and myoglobin like molecules that are found through out the animal and plant worlds. Such studies are significant for several reasons all of which stem from the sensitivity of the recombination process to global structure, site specific effects and dynamics. Ligand recombination data at ambient temperatures are often useful in establishing potential functions of a newly described hemeproteins as well as routinely being used in exploring structure-function correlations in well characterized molecules such as human hemoglobin and mammalian myoglobins. Ligand recombination in myoglobin in particular has provided biophysics with a process that has allowed for dramatic advances in understanding: i) protein dynamics, ii) the role of protein dynamics in modulating protein reactivity and iii) the interplay between the dynamical properties of the protein and the surrounding solvent. Despite the heavy focus on understanding Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptGene. Author manuscript; available in PMC 2008 August 15. Published in final edited form as:Gene. 200...

To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42°C, possibly accounting for the conservation of these residues in evolution.DNA topoisomerases (for review, see Refs. 1-7) catalyze the interconversion of different DNA topological isomers by first forming a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl on the DNA phosphodiester linkage. After strand passage through the break, religation involving nucleophilic attack of the displaced DNA hydroxyl group on the phosphotyrosine linkage takes place. Type IA and type II DNA topoisomerases are linked to the 5Ј-phosphoryl end of the cleaved DNA while type IB DNA topoisomerases are linked to the 3Ј-phosphoryl end. Mg(II) is required for the relaxation activities of both type IA and type II DNA topoisomerases but not for the type IB enzymes. The detailed catalytic mechanism of DNA cleavage and religation by topoisomerases remains to be elucidated. The mechanism of the type IA and type II topoisomerase may share similarities with other enzymes that also require Mg(II) for nucleotidyl transfer activity.Tyr-319 of Escherichia coli DNA topoisomerase I is the catalytic residue that provides the hydroxyl group for forming the covalent intermediate with DNA. The three-dimensional structure of the 67-kDa N-terminal domain of this enzyme has been determined by x-ray crystallography (8). In this structure, Tyr-319 is present in the interface between domains I and III. It has been pointed out (8) that the spatial arrangement of the three acidic residues Asp-111, Asp-113, and Glu-115 in the active site region is similar to the acidic residues that coordinate two divalent cations in the exonuclease catalytic site of Klenow fragment (9). However, the structure observed has to undergo additional conformational changes before there is sufficient space in the active site region for DNA and possibly Mg(II) to bind. A number of residues found in the active site, including Glu-9, Asp-111,...

The concept of protein dynamic states is introduced. This concept is based on (i) protein dynamics being organized hierarchically with respect to solvent slaving and (ii) which tier of dynamics is operative over the time window of a given measurement. The protein dynamic state concept is used to analyze the kinetic phases derived from the recombination of carbon monoxide to sol-gel-encapsulated human adult hemoglobin (HbA) and select recombinant mutants. The temperature-dependent measurements are made under very high viscosity conditions obtained by bathing the samples in an excess of glycerol. The results are consistent with a given tier of solvent slaved dynamics becoming operative at a time delay (with respect to the onset of the measurement) that is primarily solvent- and temperature-dependent. However, the functional consequences of the dynamics are protein- and conformation-specific. The kinetic traces from both equilibrium populations and trapped allosteric intermediates show a consistent progression that exposes the role of both conformation and hydration in the control of reactivity. Iron-zinc symmetric hybrid forms of HbA are used to show the dramatic difference between the kinetic patterns for T state alpha and beta subunits. The overall results support a model for allostery in HbA in which the ligand-binding-induced transition from the deoxy T state to the high -affinity R state proceeds through a progression of T state intermediates.

Nitrite reductase activity of deoxyhemoglobin (HbA) in the red blood cell has been proposed as a non-nitric-oxide synthase source of deliverable nitric oxide (NO) within the vasculature. An essential element in this scheme is the dependence of this reaction on the quaternary/tertiary structure of HbA. In the present work sol-gel encapsulation is used to trap and stabilize deoxy-HbA in either the T or R quaternary state, thus allowing for the clear-cut monitoring of nitrite reductase activity as a function of quaternary state with and without effectors. The results indicate that reaction is not only R-T-dependent but also heterotropic effector-dependent within a given quaternary state. The use of the maximum entropy method to analyze carbon monoxide (CO) recombination kinetics from fully and partially liganded sol-gel-encapsulated T-state species provides a framework for understanding effector modulation of T-state reactivity by influencing the distribution of high and low reactivity T-state conformations.The physiological role of the nitrite ion is currently attracting considerable research interest arising primarily from observations that indicate the anion can function as a non-nitric-oxide synthase source of nitric oxide (NO) (1-6). It has also been suggested that hemoglobin (Hb) 2 within red blood cells may be a source of deliverable nitrite-derived NO, generated from Hb nitrite reductase activity (deoxy-Hb ϩ nitrite 3 met-Hb ϩ NO) (6 -10). Such a mechanism also has clear implications for the vasoactivity of acellular hemoglobin-based blood substitutes. Although there are studies that support the physiological role of a hemoglobin-based source of nitrite-derived NO, there are still fundamental mechanistic questions that remain unanswered including the key issue of how NO, once bound to a ferrous heme, can be efficiently delivered (11, 12).Studies show that there is a relationship between the hemoglobin P50 and nitrite reductase activity (2, 3, 13, 14) as well as S-nitrosothiol synthase function (14). This result has led to the hypotheses that red blood cell/Hb enzymatic pathways can modulate blood flow and play a role in regulating hypoxic vasodilation (3, 14). The results also imply a relationship between the conformational properties of Hb and nitrite reductase activity (2, 5, 6, 15) and S-nitrosothiol synthase function (14). Direct measurements of the nitrite reductase activity of deoxy-Hb as a function of added allosteric effectors and mutagenic/chemical modifications support the concept of conformational control of Hb nitrite reductase reactivity with the T-state conformation having reduced reactivity compared with R-state conformations of Hb (5, 6). Two limitations of these studies are the inability to stabilize and compare the T and R-state forms of deoxy-HbA (human adult hemoglobin) without mutagenic or chemical modification and the complexity of the reductase reaction in solution due to autoacceleration arising from the allosteric nature of Hb reactivity.The present study directly addresses the qu...

Escherichia coli DNA topoisomerase I is the best characterized bacterial type I DNA topoisomerases (for review, see Refs. 1 and 2). Its major function in vivo is the removal of negative supercoils from DNA (3, 4). In vitro, such relaxation activity requires that Mg(II) be present in the reaction mixture (3, 5).When other divalent ions were tested (5), Ca(II) could partly replace the Mg(II) in the relaxation of negatively supercoiled DNA while the presence of other divalent ions, such as Mn(II), did not support the relaxation of negatively supercoiled DNA by the enzyme. Moreover, the presence of 2 mM Mn(II) had an inhibitory effect when co-incubated with the enzyme and 2 mM Mg(II) (5). These results suggested that there are specific interactions between Mg(II) and the enzyme or enzyme⅐DNA complex.The three-dimensional structure of the 67-kDa N-terminal fragment of E. coli DNA topoisomerase I has been determined by x-ray crystallography (6). It was noted (6) that near the active site nucleophile Tyr-319, there are three acidic residues, Asp-111, Asp-113, and Glu-115 arranged similarly to the three acidic residues known to coordinate two divalent ions in Klenow fragment (7). According to the models proposed for DNA polymerase mechanism, these coordinated divalent ions are essential for the nucleotidyl transfer catalytic activity (8, 9) of DNA polymerases. However, divalent ions were not present in the topoisomerase I crystal structure (6). It is also known that Mg(II) is not required for DNA cleavage by topoisomerase and formation of the covalent protein-DNA intermediate (10) although Mg(II) is required for intermolecular religation to be observed (11). It remains unclear if Mg(II) interacts directly with the enzyme.The basis for the requirement of Mg(II) for relaxation activity needs to be elucidated to fully understand the enzyme mechanism. There are several possible roles for Mg(II) in the relaxation of negatively supercoiled DNA by E. coli DNA topoisomerase I. For a direct role in catalysis, one or more Mg(II) bound at the active site may activate the Tyr-319 hydroxyl nucleophile and stabilize the DNA 3ЈOH-leaving group during the DNA strand cleavage step, and/or they may activate the DNA 3ЈOH as the attacking nucleophile and stabilze the Tyr-319 hydroxyl-leaving group during the DNA religation step. More indirectly, Mg(II) coordination may place the DNA phosphates and enzyme catalytic groups in the positions required for catalysis. In addition, binding of Mg(II) may allow the enzyme to undergo the conformational changes postulated to be required for the strand passage, DNA religation and substrate release steps in the proposed mechanism of reaction (6).The fluorescence of tryptophan residues is highly dependent on their local environment and enzyme conformation. We demonstrated here that significant changes in enzyme structure could be observed in the presence of 2 mM MgCl 2 , affecting the tryptophan emission intensity. This suggests that Mg(II) binding may allow the enzyme to assume a conformation necessary ...