β93 Modified Hemoglobin: Kinetic and Conformational Consequences

Journal of Biological Chemistry

Khan

et al. 2002

Self Cite

108

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...

Section: Visible Resonance Raman Spectra a Comparison Of Sol-gelencamentioning

confidence: 80%

Section: Myoglobin (Mb)mentioning

confidence: 99%

Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin

Journal of Biological Chemistry

Khan

et al. 2002

Self Cite

108

“…The hygroscopic nature of glycerol makes it difficult to prepare glycerol bathed samples that are all identical with respect to residual water content. Since the glycerol/water ratio has been shown to have clear effect on the relative amplitudes and onset of the kinetic phases (Khan et al, 2000;Khan et al, 2001;Sottini et al, 2004;Sottini et al, 2005a;Sottini et al, 2005b;Sottini et al, 2005c), it is possible that anticipated small variations in water content in different glycerol bathed sol-gel samples may influence the time points for the onset of the C and D state dynamics. There are also clear indications that the templated environment surrounding the encapsulated protein differs from that of bulk solvent in the pores of sol-gel that link the interior to the exterior of the sol-gel (Massari et al, 2006).…”

Section: Examples Illustrating the New Formalismmentioning

confidence: 99%

Ligand recombination and a hierarchy of solvent slaved dynamics: The origin of kinetic phases in hemeproteins

Roche

et al. 2007

Gene

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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...

“…The geminate yield and the solvent phase recombination rates (under the pseudo first order conditions of having a large excess of CO) for sol-gel-encapsulated COHb samples are highly responsive to both the quaternary and tertiary status of the sample (19 -21, 26, 29, 30). The CO recombination traces of samples of sol-gel-encapsulated carbonmonoxy Hbs were generated at 3.5 and 25°C using a previously described protocol in which a 7-ns 532-nm excitation pulse (10 Hz) was used to photodissociate the CO from the heme and the attenuated 442 nm CW output from a HeCd laser was used as the probe of the recombination over several decades in time (19,27,29). The evolution of T-state protocol samples was followed over a period of weeks to monitor the evolution of the kinetic traces as a function of added effectors.…”

Section: Sample Characterizationmentioning

confidence: 99%

Nitrite Reductase Activity of Sol-Gel-encapsulated Deoxyhemoglobin

Roche

Journal of Biological Chemistry

et al. 2006

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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...