Biomedical subjects

J T Sage

Publications and source records attributed to J T Sage.

18 recordsLinked to original sources

PubMed2001

Long-range reactive dynamics in myoglobin.

We report the complete vibrational spectrum of the probe nucleus 57Fe at the oxygen-binding site of the protein myoglobin. The Fe-pyrrole nitrogen stretching modes of the heme group, identified here, probe asymmetric interactions with the protein environment. Collective oscillations of the polypeptide, rather than localized heme vibrations, dominate the low frequency region. We conclude that the heme "doming" mode is significantly delocalized, so that distant sites respond to oxygen binding on vibrational time scales. This has ramifications for understanding long-range interactions in biomolecules, such as those that mediate cooperativity in allosteric proteins.

Animals↗

PubMed2001

Water penetration and binding to ferric myoglobin.

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.

Amino Acid Substitution↗

PubMed1998

Infrared crystallographic investigation of T-state hemoglobin.

Polarized IR measurements on single crystals of human hemoglobin, grown under low salt conditions that stabilize the T quaternary structure, allow spectral features to be associated with individual sites within the molecule. Differences between the a- and c-polarized IR spectra in the sulfhydryl stretching region distinguish the contributions of individual Cys residues to the S-H band and lead to an evaluation of possible H-bonding partners for Cys beta-112. Successful modelling of both crystal and solution S-H spectra with component bands having identical frequencies and bandwidths, supports the use of the X-ray structure as a model for the low affinity T-state in solution. Polarization analysis of a crystal partially saturated with CO reveals comparable occupancy of the alpha- and beta-hemes. In the T-state crystal, the C-O bands are broader and lower in frequency than in the R-state solution, and 20% of the CO-ligated beta-subunits adopt an alternate conformation with a 1967 cm(-1) C-O frequency. The latter observation reflects an energetically significant disruption of the distal heme pocket upon CO binding to beta-hemes in the low affinity T-state.

Adult↗

PubMed1997

Structural characterization of the myoglobin active site using infrared crystallography.

We use polarized IR absorption on single crystals to determine the orientation of carbon monoxide bound at the active site of myoglobin, and conclude that the C-O bond lies approximately 7 degrees from the normal to the mean plane of the heme. This result disagrees with much larger angular displacements reported in structural models derived from X-ray and neutron diffraction measurements. The insensitivity of the IR-derived orientation to changes in pH or crystal packing contrasts with the wide variations in CO orientation among diffraction-based models and suggests that the latter are in error. The small energies required to displace the C-O bond 7 degrees from its energetically preferred upright geometry suggest that distortion of the surrounding protein, rather than the relatively undeformable Fe-C-O unit, is the main steric mechanism inhibiting CO binding to myoglobin.

Animals↗

PubMed1997

Spectroscopic effects of polarity and hydration in the distal heme pocket of deoxymyoglobin.

Distal pocket mutations at the E7 position (His64) of sperm whale deoxymyoglobin (deoxyMb) are used as a probe of distal pocket polarity and hydration. Changes of two key spectroscopic markers, the Fe-His(F8) stretch in the resonance Raman spectrum and the position of band III in the absorption spectrum, are monitored as the His64Tyr, His64Phe, His64Leu, and His64Gly mutations alter the distal heme pocket environment. The Fe-His vibration for the Phe, Leu, and Gly mutants is shifted to a lower frequency by 1-2 cm-1 relative to the Tyr mutant, wild type (WT), and native deoxyMb. Band III shifts to the red by approximately 4 nm ( approximately 70 cm-1) relative to WT and native deoxyMb for all the His64 mutants examined in this work. We correlate the small shift in the Fe-His frequency to the local electrostatic environment directly above the heme iron, affected by the presence of a localized water molecule in the heme pocket that is hydrogen-bonded to the E7 residue. The position of band III is roughly correlated to the displacement of the iron from the heme plane; however, the relatively large scatter in this correlation, as well as its dependence on distal pocket mutations, suggests that the heme pocket environment, particularly the E7 residue, also affects the energy of this transition.

Animals↗

PubMed1996

Probing heme protein conformational equilibration rates with kinetic selection.

Double-pulse flash photolysis experiments on solutions of carbonmonoxymyoglobin (MbCO) are used to determine the time scale for protein conformational averaging. The interconversion times for transitions between the "open" and "closed" subpopulations of MbCO are found to be 10(-6)-10(-4)s, depending on solvent composition and temperature. In aqueous solution at 273 K, the interconversion rate is found to be 1.4 x 10(6)s. Since the interconversion rate is comparable to or slower than the geminate rebinding rate, we describe the geminate phase of the kinetics as a superposition of contributions from the open and closed states. Although geminate kinetics remain intrinsically nonexponential for both open and closed states near room temperature, we find that substates within these two subpopulations interconvert more rapidly than the geminate rebinding. These observations cannot be explained by a superposition of contributions from a quasicontinuous conformational distribution (Steinbach et al., 1991) and are probably due to the long-time tail of the relaxation of the protein (Tian et al., 1992). Bimolecular rebinding takes place at a statistically averaged rate, since the interconversion and relaxation rates are faster than the bimolecular kinetics. The geminate and bimolecular kinetics are analyzed quantitatively as a function of pH using this approach and the spectroscopically determined populations of the open and closed states. The analysis accounts for the observed kinetics and also successfully predicts the kinetic response observed in the double-pulse experiments. In aqueous solution at 273 K, the geminate amplitudes and rates are found to be I(0)g = 32% and k(0)g = 1.3 x 10(7)s(-1) for the open state and I(1)g = 9.3% and k(1)g = 1.4 x 10(6)s(-1) for the closed state. In 75% glycerol solution at 264 K, the dominant component of the geminate rebinding is characterized by I(0)g1 = 89% and k(0)g1 = 3.1 x 10(6)s(-1) for the open state and I(1)g1 = 26% and k(1)g1 = 3.1 x 10(6)s(-1) for the closed state. The fact that the interconversion rate is comparable to the geminate rate of the closed state in aqueous solution is consistent with the idea that the open state provides an important pathway for ligand escape from (or entry to) the heme pocket (Tian et al., 1993). The increased viscosity of 75% glycerol solution delays the closed--> open interconversion until the end of the geminate phase, which forces the ligand to find alternative pathways to the solution. This observation, in conjunction with the near equivalence of the geminate rates for the open and closed states in 75% glycerol solution, suggests that the solvent composition fundamentally alters the protein-ligand dynamics.

Animals↗

PubMed1994

Observation of coherent reaction dynamics in heme proteins.

Femtosecond laser pulses, resonant with Soret band of the nitric oxide complex of myoglobin (MbNO), were used to probe coherent, low-frequency nuclear motion of the heme group after photolysis. Distinct oscillations with periods of 430 and 150 femtoseconds were observed and are attributed to heme doming and iron-histidine motion, respectively. These results verify that the nuclear motion of the heme is strongly coupled to the ligand binding reaction and demonstrate that such motion is not determined by overdamped (diffusive) dynamics. The relative phases and frequencies of the nuclear motion of the photoproduct suggest that the coherence arises from impulsive electronic forces associated with the spin-state change of the heme iron atom and the depopulation of its dz2 orbital during the bond-breaking event.

Histidine↗

PubMed1993

Quantitative structural comparisons of heme protein crystals and solutions using resonance Raman spectroscopy.

Resonance Raman difference spectra have been used to compare crystal and solution samples of metmyoglobin (metMb), deoxymyoglobin (deoxyMb), and cytochrome P450. At pH 6.0, the frequency shifts of the heme core size sensitive bands v2, v3, and v4 were determined to be less than 0.3, 1.0, and 0.3 cm-1, respectively, for metMb and to be less than 1.0, 1.0, and 0.3 cm-1, respectively, for deoxyMb. This shows that the heme core size differences between the crystal and solution conformations are less than 0.002 A for metMb and less than 0.003 A for deoxyMb. These results disagree with a recent extended X-ray absorption fine structure study [Zhang, K., Chance, B., Reddy, K. S., Ayene, I., Stern, E. A., & Bunker, G. (1991) Biochemistry 30, 9116-9120] which claims that a 0.05-A difference exists in the average iron-ligand distance between the crystalline and solution forms of metMb at pH 6.5. At pH 8.5, metMb solution samples change gradually from a predominantly high-spin to a predominantly low-spin species as the ammonium sulfate concentration is increased to the level found in the crystal mother liquor. No Raman frequency shifts are found between the crystal and solution forms of metMb at pH 8.5 when the ammonium sulfate concentrations are equal. On the other hand, for deoxyMb, we find a significant alteration in the 220/240-cm-1 line shape and relative intensities, suggesting that some histidine-heme perturbation takes place upon crystallization.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Sulfate↗

PubMed1993

Investigations of ligand association and dissociation rates in the "open" and "closed" states of myoglobin.

Kinetic and Raman spectroscopic studies are combined to analyze ligand association and dissociation rates as a function of pH in aqueous solutions of myoglobin. A double-pulse flash photolysis protocol is used to kinetically select a rapidly rebinding (open pocket) fraction of the myoglobin ensemble and determine the timescale for averaging (approximately 1 to 10 microseconds) between the "open" and "closed" distal pocket protein conformations. Since this timescale is fast compared to the rate of ligand migration from the solution to the heme pocket (approximately 10(-4)s), a time-averaged population analysis, rather than a superposition of states, can be used to describe the ligand association and dissociation kinetics. Raman spectroscopy provides the relative populations of the open and closed distal pocket states as a function of pH which, in parallel with kinetics measurements, are used to determine the rates for ligand association and dissociation specific to these states. In aqueous solution at 293 K (1 mM CO) we find kon0 = 5.6 x 10(3) s-1, koff0 = 8.5 x 10(-2) s-1 for the open state and kon1 = 5.0 x 10(2) s-1, koff1 = 1.3 x 10(-2) s-1 for the closed state. The order of magnitude increase in the dissociation and association rates of the open form suggests that it may play a significant role in the ligand binding process, even though it comprises only approximately 5% of the time-averaged population at pH 7. For oxygen binding at 293 K (1.36 mM O2) we find kon0 = 4.6 x 10(4) s-1, koff0 approximately 10(4 +/- 2) s-1 for the open state and kon1 = 2.0 x 10(4) s-1, koff1 = 13 s-1 for the closed state. The dramatic increase in the dissociation rate of the open form is probably due to the loss of the hydrogen bond with the distal histidine, which stabilizes the bound O2 in the closed state. Overall, these results demonstrate that the open conformation plays a significant role in determining the ligand association and dissociation rates and suggest that environmentally induced modulations of the open population could be used as a biomolecular control mechanism for the uptake and delivery of oxygen in muscle cells.

Carbon Monoxide↗

PubMed1992

Conformational interconversion in protein crystals.

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic Fe-CO Raman modes at 508 and 491 cm-1 and is thought to involve a localized distal pocket transition from a "closed" conformation at pH 7 to a more "open" conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm-1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates (k+/-) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k+/- much greater than 10(3) s-1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k+/- approximately 10(3) to 10(4) s-1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm-1 Fe-CO modes.

Hydrogen-Ion Concentration↗

PubMed1992

Low pH myoglobin photoproducts.

Recently, there has been interest in determining the conditions under which the iron-histidine bond ruptures in myoglobin at low pH, so that the effect of proximal heme ligation can be studied. A 220-cm-1 Raman mode, assigned to iron-histidine stretching, is clearly visible after photolysis of aqueous MbCO samples below pH4 at room temperature (Sage et al. Biochemistry. 30:1237-1247). In contrast, Iben et al. (Biophys. J. 59:908-919) do not observe this mode upon photolysis of a pH3 MbCO sample in a glycerol/water glass at low temperature. In order to account for both the low temperature and the room temperature experiments, Iben et al. suggest a scheme involving an unusual protonation state of the proximal histidine. Here, we discuss some inconsistencies in their explanation of the room temperature results and offer instead a simple modification of an earlier model. In addition, circular dichroism data are presented that indicate partial unfolding of MbCO in aqueous solution below pH4, and raise questions about the claim of Iben et al. that MbCO remains folded in 75% glycerol at pH3.

Biophysical Phenomena↗

PubMed1991

Spectroscopic studies of myoglobin at low pH: heme structure and ligation.

We explore heme structure and ligation subsequent to a low-pH conformational transition in sperm whale myoglobin. Below pH 4.0, the iron-histidine bond breaks in metMb and deoxyMb. In MbCO, the majority of the iron-histidine bonds remain intact down to pH 2.6; however, the observation of a weak Fe-CO mode at 526 cm-1 indicates that a small fraction of the sample has the histidine replaced by a weak ligand, possibly water. The existence of a sterically hindered CO subpopulation in MbCO and the continued association of the four-coordinate heme with the protein in deoxyMb suggest that the heme pocket remains at least partially intact in the acid-induced conformation. The global pH-dependent conformational change described here is clearly distinguished from the local "closed" to "open" transition described previously in MbCO [Morikis et al. (1989) Biochemistry 28, 4791-4800]. Further observations of the four-coordinate heme state yield insights on the mechanism of heme photoreduction and the assignment of the 760-nm band in deoxyMb.

Animals↗

PubMed1991

Spectroscopic studies of myoglobin at low pH: heme ligation kinetics.

On the basis of the characterization of heme structure and ligation in equilibrium, we explore both proximal and distal ligation kinetics of myoglobin below pH 4. Upon photolysis of MbCO, a significant five-coordinate heme population is observed, with an intact iron-histidine bond that persists on the time scale of CO rebinding. Incomplete CO photolysis is attributed to a rapidly exchanging minority population of four-coordinate hemes, which leads to fast (greater than 10(10) s-1) geminate recombination. The possible relevance of such a mechanism at pH 7 is also noted. Using a novel experimental protocol, we observe the resonance Raman spectrum of partially photolyzed MbCO as a function of continuous wave illumination time (tau). Under extended illumination (tau approximately 35 ms at pH 3.4), there is a loss of intensity in the nu 4 region of the Raman spectrum and the iron-histidine mode is bleached from the spectrum of the five-coordinate photoproduct. In the Fe-CO stretching region of the CO-bound fraction, the intensity of the 526-cm-1 mode increases with tau at the expense of the 491-cm-1 mode. These changes are interpreted as being due to replacement of the proximal histidine ligand under continuous illumination. Complete relaxation to the pure four-coordinate deoxy heme structure observed in equilibrium is not observed even as tau----infinity, presumably since CO rebinding leads to acidification of the iron and its complexation with histidine. We propose a kinetic model to account for our results and discuss the implications for previous low-pH kinetics measurements.

Animals↗

PubMed1988

Nitric oxide adducts of the binuclear iron site of hemerythrin: spectroscopy and reactivity.

Nitric oxide forms adducts with the binuclear iron site of hemerythrin (Hr) at [Fe(II),Fe(II)]deoxy and [Fe(II),Fe(III)]semimet oxidation levels. With deoxyHr our results establish that (i) NO binds reversibly, forming a complex which we label deoxyHrNO, (ii) NO forms a similar but distinct complex in the presence of fluoride, which we label deoxyHrFNO, (iii) NO is directly coordinated to one iron atom of the binuclear pair in these adducts, most likely in a bent end-on fashion, and (iv) the iron atoms in the binuclear sites of both deoxyHrNO and deoxyHrFNO are antiferromagnetically coupled, thereby generating unique electron paramagnetic resonance (EPR) detectable species. The novel EPR signal of deoxyHrNO (deoxyHrFNO) with g[[ = 2.77 (2.58) and g = 1.84 (1.80) is explained by the magnetic interaction of the Fe(II) (S' = 2) and [FeNO]7 (S = 3/2) centers observed by Mössbauer spectroscopy. Antiferromagnetic coupling leads to a ground state of Seff = 1/2. Analysis of the EPR parameters using the isotropic spin-exchange Hamiltonian, Hex = 2JS3/2.S2, and including zero-field splitting leads to a coupling constant, -J approximately 23 cm-1, for deoxyHrNO. The resonance Raman spectrum of deoxyHrNO shows features at 433 and 421 cm-1 that shift downward with 15N16O and that are assigned to stretching and bending modes, respectively, of the [FeNO]7 unit. Sensitivity of the bending mode to D2O suggests that bound NO participates in hydrogen bonding. We propose that the terminal oxygen atom of NO is hydrogen bonded to the proton of the mu-hydroxo bridge in the Fe-(OH)-Fe unit. A bent Fe-N-O geometry is supported by spectroscopic and structural comparisons to synthetic complexes and is consistent with a limiting [FeII,FeIIINO-] formulation for deoxyHrNO. Reversibility of NO binding to deoxyHr is demonstrated by bleaching of the optical and EPR spectra of deoxyHrNO upon additions of excess N3- or CNO-. DeoxyHrNO undergoes autoxidation under anaerobic conditions over the course of several hours. The product of this autoxidation appears to be an EPR-silent NO adduct of semimetHr. The formal one-electron oxidations of the binuclear iron site of deoxyHr by NO and by HNO2 can conceivably occur with no net change in charge on the iron site. In contrast, autoxidation of oxy- to metHr requires a change in net charge on the iron site, which may provide a kinetic barrier.

Animals↗

PubMed1986

The interaction of phosphate with uteroferrin. Characterization of a reduced uteroferrin-phosphate complex.

The interaction of phosphate with reduced uteroferrin has been re-examined in light of disagreements on the oxidation state of the binuclear iron cluster (Keough, D. T., Beck, J. L., de Jersey, J., and Zerner, B. (1982) Biochem. Biophys. Res. Commun. 108, 1643-1648; Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756). Our results based on Mossbauer observations and the kinetics of spectral change and activity loss show clearly that phosphate binds to reduced uteroferrin to form a reduced uteroferrin-phosphate complex. This complex exhibits a pair of quadrupole doublets at 119 K with parameters typical of a high spin ferric and a high spin ferrous center, respectively, but distinct from those of the native reduced enzyme. The reduced phosphate complex exhibits a pH-dependent visible absorption maximum ranging from 530 to 561 nm. In air, the reduced phosphate complex converts to the oxidized phosphate complex with a first order rate constant of 4 X 10(-3) min-1, as monitored by spectral changes and loss of enzyme activity.

Acid Phosphatase↗

PubMed1984

Mössbauer and electron paramagnetic resonance studies of horseradish peroxidase and its catalytic intermediates.

We report Mössbauer and EPR measurements on horseradish peroxidase in the native state and the reaction intermediates with peroxide and chlorite. A detailed analysis of the electronic state of the heme iron is given, and comparisons are drawn with related systems. The native enzyme is high-spin ferric and thus has three Kramers doublets. The unusual magnetic properties of the ground doublet and the large energy of the second, (E2-E1)/k approximately equal to 41 K, and third doublet, (E3-E1)/k greater than or equal to 170 K, can be modeled with a quartet admixture of approximately 11% to the spin sextet. All evidence suggests a ferryl, OFeIV, state of the heme iron in compounds I and II and related complexes. The small isomer shift, delta Fe approximately equal to 0.06 mm/s, the (positive) quadrupole splitting, delta EQ approximately equal to 1.4 mm/s, the spin S = 1, and the large positive zero field splitting, D/k approximately equal to 35 K, are all characteristic of the ferryl state. In the green compound I the iron weakly couples to a porphyrin radical with spin S' = 1/2. A phenomenological model with a weak exchange interaction S . J . S', magnitude of less than or equal to 0.1 D, reproduces all Mössbauer and EPR data of compound I, but the structural origin of the exchange and its apparent distribution require further study. Reaction of horseradish peroxidase with chlorite leads to compound X with delta Fe = 0.07 mm/s and delta EQ = 1.53 mm/s, values that are closest to those of compound II. The diamagnetism of compound III and its Mössbauer parameters delta Fe = 0.23 mm/s and delta EQ = -2.31 mm/s at 4.2 K clearly identify it as an oxyheme adduct.

Electron Spin Resonance Spectroscopy↗

PubMed1983

Mössbauer and EPR study of the binuclear iron centre in purple acid phosphatase.

Mössbauer spectra have been determined on 57Fe-enriched samples of both pink (reduced) and purple (oxidized) forms of pig allantoic acid phosphatase (EC 3.1.3.2), and EPR spectra on corresponding unenriched samples. The spectra show unambiguously that both forms of the enzyme contain two distinct, antiferromagnetically coupled, high-spin iron atoms: a ferrous-ferric ion pair in the pink, reduced form, and a pair of ferric ions in the purple, oxidized form.

Acid Phosphatase↗

PubMed1983

Semi-met oxidation level of chalcogenide derivatives of methemerythrin. Mössbauer and EPR studies.

Conclusive evidence is presented for an S = 1/2 spincoupled pair of high spin ferric and ferrous ions in the major reaction product of sulfide with the met form of the non-heme iron oxygen-carrying protein hemerythrin. Evidence for an analogous selenide derivative is also reported. Mössbauer and EPR spectroscopy establish (a) the charge and spin states of the individual iron atoms in sulfidehemerythrin as Fe(III), S = 5/2, and Fe(II), S = 2, and (b) the existence of an antiferromagnetic exchange interaction that couples the two spins to a resultant spin S = 1/2. The combined Mössbauer and EPR data confirm the correctness of the formulation first proposed for semi-methemerythrin by Harrington, P.C., de Waal, D.J.A., and Wilkins, R.G. ((1978) Arch. Biochem. Biophys. 191, 444-451) and furthermore show that a majority of the iron centers in the protein can be stabilized at this oxidation level. The results also demonstrate a new route to semi-methemerythrin. A titration of methemerythrin with selenide indicates that this derivative forms by a two step process consisting of first, reduction to the semi-met oxidation level by selenide and second, binding of selenide to either one or both irons.

Animals↗