Carbonmonoxy Myoglobin Research Articles

X-ray fluorescence holography of biological metal sites: Application to myoglobin

X-ray fluorescence holography (XFH) is a relatively new technique capable of providing unique three-dimensional structural information around specific atoms that act as a light source in crystalline samples. So far, XFH has typically been applied to inorganic materials such as dopants in metals and semiconductors. Here, we investigate the possibility of using XFH to visualize the metal active site in sperm whale myoglobin (Mb), a monomeric oxygen storage heme protein. We demonstrate that the atomic images reconstructed from the hologram data of crystals of carbonmonoxy myoglobin (MbCO) are moderately consistent with the crystal structure, which is also determined in this study by X-ray crystallography in the near-atomic resolution, as well as simulation results. These results open up a new avenue for the application of XFH to local atomic and electronic structure imaging of metal-sites in biomolecules.

Ultrafast Structural Changes Decomposed from Serial Crystallographic Data.

Direct visualization of electronic and molecular events during biochemical reactions is essential to mechanistic insights. This Letter presents an in-depth analysis of the serial crystallographic data sets collected by Barends and Schlichting et al. ( Science 2015 , 350 , 445 ) that probe the ligand photodissociation in carbonmonoxy myoglobin. This analysis reveals electron density changes caused by the formation of high-spin 3d atomic orbitals of the heme iron upon photolysis and their dynamic behaviors within the first few picoseconds. The heme iron is found popping out of and recoiling back into the heme plane in succession. These findings provide long-awaited visual validations for previous works using ultrafast spectroscopy and molecular dynamics simulations. Electron density variations are also found largely in the solvent during the first period of a low-frequency oscillation. This work demonstrates the importance of the analytical methods in detecting and isolating weak, transient signals of electronic changes arising from chemical reactions.

Time-Resolved Linear Dichroism Measurements of Carbonmonoxy Myoglobin as a Probe of the Microviscosity in Crowded Environments.

The distribution of viscosities in living cells is heterogeneous because of the different sizes and natures of macromolecular components. When thinking about protein folding/function processes in such an environment, the relevant (micro)viscosity at the micrometer length scale is necessarily distinguished from the bulk (macro)viscosity. The concentration dependencies of microviscosities are determined by a number of factors, such as electrostatic interactions, van der Waals forces, and excluded volume effects. To explore such factors, the rotational diffusion time of myoglobin in the presence of varying concentrations of macromolecules that differ in molecular weight (dextran 6000, 10 000, and 70 000), shape (dextran versus Ficoll), size, and surface charge is measured with time-resolved linear dichroism spectroscopy. The results of these studies offer simple empirically determined linear and exponential functions useful for predicting microviscosities as a function of concentration for these macromolecular crowders that are typically used to study crowding effects on protein folding. To understand how relevant these microviscosity measurements are to intracellular environments, the TRLD results are discussed in the context of studies that measure viscosity in cells.

Observing heme doming in myoglobin with femtosecond X-ray absorption spectroscopy.

We report time-resolved X-ray absorption measurements after photolysis of carbonmonoxy myoglobin performed at the LCLS X-ray free electron laser with nearly 100 fs (FWHM) time resolution. Data at the Fe K-edge reveal that the photoinduced structural changes at the heme occur in two steps, with a faster (∼70 fs) relaxation preceding a slower (∼400 fs) one. We tentatively attribute the first relaxation to a structural rearrangement induced by photolysis involving essentially only the heme chromophore and the second relaxation to a residual Fe motion out of the heme plane that is coupled to the displacement of myoglobin F-helix.

Structure of A0, A1 and A3 conformational substates of carbonmonoxy myoglobin

Infrared absorption spectra of carbon monoxide molecule coordinated by the heme iron of carbonmonoxy heme proteins are widely used to study their structure and dynamics. In this paper we use results of X-ray study of carbonmonoxy myoglobin to elucidate the structures of spectroscopically observed conformational substates of this protein. It is shown that A3 substate corresponds to the structure with water molecule hydrogen bonded to the distal histidine, whereas in the A1 conformation this molecule is absent. We also show that redistribution of electronic density of the distal histidine and the water molecule as a result of their interaction must be taken into account when predicting their positions in the heme pocket. 1. Background Relationship between the dynamics of biological molecules their structure and function is an important problem of modern chemistry and physics. Heme proteins play a special role in these studies because their active center, heme, can be studied by virtually all spectroscopic techniques. In particular, kinetic measurements of CO recombination after photolysis of carbonmonoxy myoglobin (Mb(CO)) lead to the view of conformational substates (CSS) and energy barriers (2,3,5). Population of different CSSs is very important for the protein function and causes strongly anharmonic motion of proteins and their very specific function (1,4). It was shown that in Mb(CO) there are at least three structurally different CSSs, which correspond to three peaks in the infrared absorption spectrum of the carbon monoxide coordinated by the heme iron at: νCO ≈ 1966 cm −1 , 1945 cm −1 and 1933 cm −1 (11). These three peaks were ascribed to three conformational substates: A0 ,A 1 and A3, respectively. The variation in the CO peak position was shown to be caused by electrostatic interaction of the heme with the protein electric filed (7-9,13). The A0 CSS was ascribed to a conformation with the distal histidine located out of the heme pocket (7,8,12,13,16). The structures of the A1 and A3 CSSs were discussed intensively, but all attempts to elucidate these structures using different theoretical approaches, including molecular dynamics (10,14), failed. At the same time recently the crystal structure of Mb(CO) (15) was revised (PDB entrance 1a6g), the latest data clearly showed presence of three CSSs.

Palmitate interaction with physiological states of myoglobin

BackgroundPrevious studies have shown that palmitate (PA) can bind specifically and non-specifically to Fe(III) MbCN. The present study has observed PA interaction with physiological states of Fe(II) Mb, and the observations support the hypothesis that Mb may have a potential role in facilitating intracellular fatty acid transport. Methods1H NMR spectra measurements of the Mb signal during PA titration show signal changes consistent with specific and non-specific binding. ResultsPalmitate (PA) interacts differently with physiological states of Mb. Deoxy Mb does not interact specifically or non-specifically with PA, while the carbonmonoxy myoglobin (MbCO) interaction with PA decreases the intensity of selective signals and produces a 0.15ppm upfield shift of the PA methylene peak. The selective signal change upon PA titration provides a basis to determine an apparent PA binding constant, which serves to create a model comparing the competitive PA binding and facilitated fatty acid transport of Mb and fatty acid binding protein (FABP). ConclusionsGiven contrasting PA interaction of ligated vs. unligated Mb, the cellular fatty acid binding protein (FABP) and Mb concentration in the cell, the reported cellular diffusion coefficients, the PA dissociation constants from ligated Mb and FABP, a fatty acid flux model suggests that Mb can compete with FABP transporting cellular fatty acid. General significanceUnder oxygenated conditions and continuous energy demand, Mb dependent fatty acid transport could influence the cell's preference for carbohydrate or fatty acid as a fuel source and regulate fatty acid metabolism.

Clarification of the role of protein in carbonmonoxy myoglobin by investigating electronic states

This article reports the proton tautomerization effects of distal histidine residues in carbonmonoxy myoglobin according to the density functional calculations of the whole protein. The electron eigenstates and electrostatic potential (ESP) distributed around heme and its pocket vary significantly depending on the protonation positions of the distal histidine residues. To investigate the range over which the electronic structures are affected by the proton tautomerization, the quantum mechanics/molecular mechanics (QM/MM) method is applied to probe the QM size to reproduce the atomic partial charges and ESP around the active center. Consequently, we show that these properties converged for the 300 pm QM/MM system in this study. During the analysis, we also find that amino residues such as Phe43, Val68, and Phe138 interact strongly with heme through orbital mixing, indicating that the protein is a medium not only interacting with the reaction center, but also buffering on electrons. © 2013 Wiley Periodicals, Inc.

Incipient structural and vibrational relaxation process of photolyzed carbonmonoxy myoglobin: statistical analysis by perturbation ensemble molecular dynamics method

The incipient structural and vibrational energy relaxation process of photolyzed carbonmonoxy myoglobin was analyzed by the perturbation ensemble molecular dynamics (PEMD) method, in which many pairs of perturbed and unperturbed MD simulations are executed for ensemble-averaging to obtain statistically significant results by canceling out thermal fluctuations. First, we have shown that the experimentally reported anisotropic expansion can be detected within a picosecond after photolysis. The good agreement between the experimental and computational results indicates that the PEMD method can predict legitimately those changes driven by perturbations even if the changes might be subtle and smaller than thermal fluctuations. Second, the structural relaxation including the “clamshell rotation” in E and F helices was successfully analyzed. The high time resolution analysis has clarified the incipient structural dynamics on a subpicosecond timescale: the clamshell rotation starts at His64, Val68, and His93 following both the heme doming and the dissociated CO ligand collision. Third, the vibrational energy relaxation from the heme to the globin matrix is elucidated not only temporally but also spatially. This is the first “thorough” report of the spacetime-resolved excess kinetic energy redistribution of photolyzed MbCO in the globin matrix with a statistically significant precision, ±1 K. The incipient anisotropic vibrational relaxation occurs clearly within a picosecond in the direction perpendicular to the heme plane by the “through-bond” and “through-projectile” pathways, and the isotropic relaxation then follows by the “through-space” pathway. Finally, it is concluded that the PEMD method is a powerful tool to understand the incipient relaxation process driven by the perturbation.

Implementation of Time-Resolved Step-Scan Fourier Transform Infrared (FT-IR) Spectroscopy Using a kHz Repetition Rate Pump Laser

Time-resolved step-scan Fourier transform infrared (FT-IR) spectroscopy has been shown to be invaluable for studying excited-state structures and dynamics in both biological and inorganic systems. Despite the established utility of this method, technical challenges continue to limit the data quality and more wide ranging applications. A critical problem has been the low laser repetition rate and interferometer stepping rate (both are typically 10 Hz) used for data acquisition. Here we demonstrate significant improvement in the quality of time-resolved spectra through the use of a kHz repetition rate laser to achieve kHz excitation and data collection rates while stepping the spectrometer at 200 Hz. We have studied the metal-to-ligand charge transfer excited state of Ru(bipyridine)(3)Cl(2) in deuterated acetonitrile to test and optimize high repetition rate data collection. Comparison of different interferometer stepping rates reveals an optimum rate of 200 Hz due to minimization of long-term baseline drift. With the improved collection efficiency and signal-to-noise ratio, better assignments of the MLCT excited-state bands can be made. Using optimized parameters, carbonmonoxy myoglobin in deuterated buffer is also studied by observing the infrared signatures of carbon monoxide photolysis upon excitation of the heme. We conclude from these studies that a substantial increase in performance of ss-FT-IR instrumentation is achieved by coupling commercial infrared benches with kHz repetition rate lasers.

Structural characterization of spectroscopic substates in carbonmonoxy neuroglobin

Relating structure and spectroscopy is fundamental in characterizing the conformational dynamics and elucidating function at an atomistic level in condensed-phase environments. In particular, the combination of infrared spectroscopy and atomistic simulations has provided fundamental insight into structural assignments of spectroscopic bands. Infrared spectroscopy and Molecular Dynamics (MD) simulations on carbonmonoxy myoglobin (MbCO) were able to partially identify three major CO infrared bands (A0, A1 and A3) which are related to different conformational substates in the active site of the protein. Recently, two similar CO bands were identified from experiments in human carbonmonoxy neuroglobin (NgbCO), named N0 and N3. Time-dependent frequency changes found in the N0 band and a large variation of relaxation times for these bands made the characterization of these substates considerably more difficult compared to MbCO. In this work we discuss the structure-spectroscopy relationship for three different His64 protonation states in human and murine NgbCO using MD simulations and density functional theory (DFT) calculations. The present work assigns the N3 band to the His(epsilon)64 tautomer having its side chain in hydrogen bonding contact to CO. Frequencies of the corresponding His64H+ and His(delta)64 tautomers show characteristic contributions to the N0 band.

1I1448 摂動アンサンブルMD法による光解離MbCOの初期緩和過程の高分解能解析(ヘム蛋白質 1,第49回日本生物物理学会年会)

1I1448 摂動アンサンブルMD法による光解離MbCOの初期緩和過程の高分解能解析(ヘム蛋白質 1,第49回日本生物物理学会年会)

Structural Dynamics of Clamshell Rotation during the Incipient Relaxation Process of Photodissociated Carbonmonoxy Myoglobin: Statistical Analysis by the Perturbation Ensemble Method

The structural dynamics of the clamshell rotation of photodissociated carbonmonoxy myoglobin, which is expected to be important for hemoglobin allostery, is investigated by the perturbation ensemble method. In this method, many pairs of perturbed and unperturbed molecular dynamics trajectories are ensemble-averaged to cancel out thermal noises and to detect subtle changes. The number of MD trajectory pairs, in this work 2000 pairs, should be determined to obtain physical properties of interest with statistically meaningful precisions. The calculated structural changes after 20 ps of the photodissociation are consistent with those by time-resolved X-ray diffraction at 100 ps delay time. In the heme proximal side region including the F and H helices, both helices displaced in the proximal direction. Meanwhile, in the heme distal side region including E and A helices, both helices moved toward the heme group after photodissociation. These proximal and distal side displacements occur on a fast time scale (almost complete within 3 ps) and are consistent with the clamshell rotation. Moreover, it was found that the ensemble-averaged structural dynamics of the photodissociated MbCO is independent of the amount of initial excess vibrational energy of the heme, or the difference of excitation photon wavelength. These results provide atomistic details on the functionally important dynamics of the clamshell rotation. Application of the present methodology to Hb will give new insight into the incipient stereochemical mechanism of hemoglobin allostery.

CO Releasing Properties and Cytoprotective Effect of cis-trans- [ReII(CO)2Br2L2]n Complexes

The carbon monoxide (CO) releasing properties of a series of rhenium(II)-based complexes of general formula cis-trans-[Re(II)(CO)(2)Br(2)L(2)](n) and cis-trans-[Re(II)(CO)(2)Br(2)N[intersection]N] (where L = monodentate and N[intersection]N = bidentate ligand) are reported. Complexes evaluated in this study were obtained from direct ligand substitution reactions of the cis-[Re(II)(CO)(2)Br(4)](2-) synthon (2) recently described. (1) All molecules have been fully characterized. The solid-state structures of the cis-trans-[Re(II)(CO)(2)Br(2)L(2)] (with L = N-methylimidazole (3), benzimidazole (4) and 4-picoline (5)) and the cis-trans-[Re(II)(CO)(2)Br(2)N[intersection]N] (with N[intersection]N = 4,4'-dimethyl-2,2'-bipyridine (8) and 2,2'-dipyridylamine (11)) adducts are presented. The release of CO from the cis-trans-[Re(II)(CO)(2)Br(2)L(2)](n) complexes was assessed spectrophotometrically by measuring the conversion of deoxymyoglobin (Mb) to carbonmonoxy myoglobin (MbCO). Only compounds bearing monodentate ligands were found to liberate CO. The rate of CO release was found to be pH dependent with half-lives (t(1/2)) under physiological conditions (25 degrees C, 0.1 M phosphate buffer, and pH 7.4) varying from ca. 6-43 min. At lower pH values, the time required to fully saturate Mb with CO liberated from the metal complexes gradually decreased. Complex 2 and the cis-trans-[Re(II)(CO)(2)Br(2)(Im)(2)] adduct (with Im = imidazole (6)) show a protective action against "ischemia-reperfusion" stress of neonatal rat ventricular cardiomyocytes in culture.

Tracking ligand-migration pathways of carbonmonoxy myoglobin in crystals at cryogenic temperatures

In order to explore the ligand-migration dynamics in myoglobin induced by photodissociation, cryogenic X-ray crystallographic investigations of carbonmonoxy myoglobin crystals illuminated by continuous wave and pulsed lasers at 1-15 kHz repetition rate have been carried out. Here it is shown that this novel method, extended pulsed-laser pumping of carbonmonoxy myoglobin, promotes ligand migration in the protein matrix by crossing the glass transition temperature repeatedly, and enables the visualization of the migration pathway of the photodissociated ligands in native Mb at cryogenic temperatures. It has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino-acid residues around the cavity which result in the expansion of the cavity. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand-migration channel arising by induced fit.

Structural Assignment of Spectra by Characterization of Conformational Substates in Bound MbCO

Residue motions of the distal heme pocket and bound CO ligand of carbonmonoxy Myoglobin are studied using a combination of molecular dynamics simulations and quantum chemical methods. Using mixed quantum mechanics/molecular mechanics calculations together with sampling from molecular dynamics simulations (QM/MM(MD)), the experimentally observed spectroscopic A0 and A1 substates of the bound CO ligand are assigned to the open and closed conformation of His64 and the Hisɛ64 tautomer, respectively. Several previously proposed origins of the A3 substate, including rotamers of the doubly protonated His64H+ side chain, His64H+ inside the distal pocket, and cooperative motions with Arg45, are investigated with QM/MM(MD). However, the signatures of the calculated infrared spectra do not agree with the experimentally observed ones. For additional insight on this, extensive molecular dynamics simulations are used together with improved electrostatics for the bound ligand. A CO fluctuating charge model is developed to describe the ab initio dipole and quadrupole moments of the bound ligand. CO absorption spectra are then obtained directly from the dynamics simulations. Finally, the electrostatics of the heme pocket is examined in detail in an attempt to determine the structural origins of the observed spectroscopic A-states from MD simulations. However, contrary to related simulations for unbound CO in myoglobin, the shifts and splittings for carbonmonoxy Myoglobin are generally small and difficult to relate to structural change. This suggests that coupling of the CO motion to other degrees of freedom, such as the Fe-CO stretching and bending, is important to correctly describe the dynamics of bound CO in myoglobin.

Probing the Role of Hydration in the Unfolding Transitions of Carbonmonoxy Myoglobin and Apomyoglobin

We show that the equilibrium unfolding transition of horse carbonmonoxy myoglobin monitored by the stretching vibration of the CO ligand, a local environmental probe, is very sharp and, thus, quite different from those measured by global conformational reporters. In addition, the denatured protein exhibits an A(0)-like CO band. We hypothesize that this sharp transition reports penetration of water into the heme pocket of the protein. Parallel experiments on horse apomyoglobin, wherein an environment-sensitive fluorescent probe, nile red, was used, also reveals a similar putative hydration event. Given the importance of dehydration in protein folding and also the recent debate over the interpretation of probe-dependent unfolding transitions, these results have strong implications on the mechanism of protein folding.

Linear Response of Biomolecules To External Perturbations: Revisit Induce-fit

Atoms in proteins, viewed as interlinked hubs, communicate with each other in complying with governed physical forces. The deviations of their positions from the mean, known as fluctuations, are essential in mediating functionally relevant biological processes maintaining one's daily life. The coupled fluctuations between pairs of atoms, the fluctuation covariance, can be determined analytically by Normal Mode Analysis, or numerically by MD simulations. Our study considers how this network of atoms reacts in response to external perturbations. Effects of such perturbations are exemplified by ligand- or (another) protein-induced conformational changes as well as the appreciated structural distortion of crystalline structures from its solution conformers. We assume the response of the system has linear departure from the mean under small perturbations on the Hamiltonian at equilibrium state. The formulated linear response theories, either time-dependent or -independent [1], says that the positional change of a given atom i is the accumulative sum of fluctuation covariance ij, at unperturbed state, multiplied by the force exerted on atom j. The time-dependent response function determined from MD simulation of carbonmonoxy myoglobin is used to track time-dependent conformational changes upon photo-dissociation of CO. The consequently obtained understanding of perturbation propagation is compared with experimental results. Also, the structural distortion of X-ray-characterized ubiquitin from its solution conformer can be well explained by the induce-fit theory, in a wider sense, while population-shift does not account for such a deviation.[1] Ikeguchi M, Ueno J, Sato M, and Kidera A. (2005) Phys Rev Lett, 94, 078102.

Dynamic fluctuation of proteins watched in real time

The dynamic nature of protein function is a fundamental concept in the physics of proteins. Although the basic general ideas are well accepted most experimental evidence has an indirect nature. The detailed characterization of the dynamics is necessary for the understanding in detail. The dynamic fluctuations thought crucial for the function span an extremely broad time, starting from the picosecond regime. Recently, a few new experimental techniques emerged that permit the observation of dynamical phenomena directly. Notably, pulsed infrared (IR) spectroscopy has been applied with great success to observe structural changes with picosecond time resolution. Using two-dimensional-IR vibrational echo chemical exchange spectroscopy Ishikawa and co-workers [Ishikawa et al. (2008), Proc. Natl. Acad. Sci. U.S.A. 101, 14402-14407] managed to observe the transition between well defined conformational substrates of carbonmonoxy myoglobin directly. This is an important step in improving our insight into the details of protein function.

Slow ligand migration dynamics in carbonmonoxy myoglobin at cryogenic temperature

Slow ligand migration dynamics in carbonmonoxy myoglobin at cryogenic temperature

Mössbauer spectroscopic evidence on the heme binding to the proximal histidine in unfolded carbonmonoxy myoglobin by guanidine hydrochloride

The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Mössbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Mössbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.