Active site conformation in myoglobin as determined by X-ray absorption spectroscopy

X-ray absorption fine structure experiments were performed to study structural and dynamic aspects of the active site of various forms of myoglobin. The structures determined for deoxyMb, MbCO, and MbO2 are consistent with the structure established by X-ray absorption fine structure experiment and X-ray crystallography. The first shell of ferrous MbNO determined contains 5 nitrogens located at 2.02 A and a short NO bond length of 1.76 A. This study focuses on the change of the XAFS Debye-Waller factor with temperature, which is a measure of thermal and static disorder. It was found that the changes of Debye-Waller factor with temperature for the Mb proteins, except deoxyMb, are consistent with a simple Einstein model, in which a single frequency was assumed for the bond stretching modes. In contrast, the temperature dependence of deoxyMb cannot be fitted to the Einstein model and a large disorder was found at low temperatures, which indicates the existence of conformational substates of the active site.     

Fe-heme conformations in ferric myoglobin.

X-ray absorption near-edge structure (XANES) spectra of ferric myoglobin from horse heart have been acquired as a function of pH (between 5.3 and 11.3). At pH = 11.3 temperature-dependent spectra (between 20 and 293 K) have been collected as well. Experimental data solve three main conformations of the Fe-heme: the first, at low pH, is related to high-spin aquomet-myoglobin (Mb+OH2). The other two, at pH 11.3, are related to hydroxymet-myoglobin (Mb+OH-), and are in thermal equilibrium, corresponding to high- and low-spin Mb+OH-. The structure of the three Fe-heme conformations has been assigned according to spin-resolved multiple scattering simulations and fitting of the XANES data. The chemical transition between Mb+OH2 and high-spin Mb+OH-, and the spin transition of Mb+OH-, are accompanied by changes of the Fe coordination sphere due to its movement toward the heme plane, coupled to an increase of the axial asymmetry.     

The structural dynamics of myoglobin

Conformational fluctuations in proteins were initially invoked to explain the observation that diffusion of small ligands through the matrix is a global phenomenon. Small globular proteins contain internal cavities that play a role not only in matrix dynamics but also in controlling function, tracing a pathway for the diffusion of the ligand to and from the active site. This is the main point addressed in this Review, which presents pertinent information obtained on myoglobin (Mb). Mb, a simple globular heme protein which binds reversibly oxygen and other ligands. The bond between the heme Fe(II) and gaseous ligands can be photodissociated by a laser pulse, generating a non-equilibrium population of protein structures that relaxes on a picosecond to millisecond time range. This process is associated with migration of the ligand to internal cavities of the protein, which are known to bind xenon. Some of the results obtained by laser photolysis, molecular dynamics simulations, and X-ray diffraction of intermediate states of wild-type and mutant myoglobins are summarized. The extended relaxation of the globin moiety directly observed by Laue crystallography reflects re-equilibration among conformational substates known to play an essential role in controlling protein function.     

The Fe-heme structure of met-indoleamine 2,3-dioxygenase-2 determined by X-ray absorption fine structure

Multiple-scattering (MS) analysis of EXAFS data on met-indoleamine 2,3-dioxygenase-2 (IDO2) and analysis of XANES have provided the first direct structural information about the axial donor ligands of the iron center for this recently discovered protein. At 10 K, it exists in a low-spin bis(His) form with Fe–N_p(av) = 1.97 Å, the Fe–N_Im bond lengths of 2.11 Å and 2.05 Å, which is in equilibrium with a high-spin form at room temperature. The bond distances in the low-spin form are consistent with other low-spin hemeproteins, as is the XANES spectrum, which is closer to that of the low-spin met-Lb than that of the high-spin met-Mb. The potential physiological role of this spin equilibrium is discussed.     

Structural dynamics of myoglobin: spectroscopic and structural characterization of ligand docking sites in myoglobin mutant L29W

We have studied CO binding to the heme and CO migration among protein internal cavities after photodissociation in sperm whale carbonmonoxy myoglobin (MbCO) mutant L29W using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) and kinetic experiments at cryogenic temperatures. Photoproduct intermediates, characterized by CO at particular locations in the protein, were selectively enhanced by applying special laser illumination protocols. These studies were performed on the L29W mutant protein and a series of double mutants constructed so that bulky amino acid side chains block passageways between cavities or fill these sites. Binding of xenon was also employed as an alternative means of occluding cavities. All mutants exhibit two conformations, A(I) and A(II), with distinctly different photoproduct states and ligand binding properties. These differences arise mainly from different positions of the W29 and H64 side chains in the distal heme pocket [Ostermann, A., et al. (2000) Nature 404, 205-208]. The detailed knowledge of the interplay between protein structure, protein dynamics, and ligand migration at cryogenic temperatures allowed us to develop a dynamic model that explains the slow CO and O(2) bimolecular association observed after flash photolysis at ambient temperature.     

Low temperature X-ray investigation of structural distributions in myoglobin

The results of X-ray structure analysis of metmyoglobin at 300 K, 185 K, 165 K, 115 K and 80 K are reported. The lattice vectorsa andb decrease linearly with temperature whilec shows non-linearity above 180 K, indicating some type of phase transition. Cooling does change the myoglobin structure but only within the structural distribution as determined by individual x ² at room temperature. Two residues showed significant alternative positions for sidechains at higher temperatures while only one position is occupied at low temperatures. In the case of LEU 61 a jump between different positions of the side-chain reduces the potential barrier for the entrance of the O₂ molecule to the heme pocket.The mean square displacements, x ², of the individual residues decrease linearly with temperature in most cases, indicating a parabolic envelope for the potential responsible for motions. A separation of rotational and translational disorder of the entire molecule is discussed. Comparison with Mössbauer spectroscopy indicates that protein dynamics on a time scale faster than 10^-7 s is not simply a harmonic process. Extrapolation of the structural distributions toT=0 K shows that a large zero point distribution of the myoglobin structure exists, thus proving that there is no absolute energy minimum for one well defined conformation.Dedicated to Prof. H. Frauenfelder on his 65^th birthday     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Structural dynamics of myoglobin: ligand migration among protein cavities studied by Fourier transform infrared/temperature derivative spectroscopy.

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A(0) and A(3), which can be distinguished by their different CO bands near 1965 and 1933 cm(-1). They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C', in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C) has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at approximately 180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C', C, and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C') is severely restricted by introduction of the bulky Phe side chain at position 107.     

X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2

Human ABH2 repairs DNA lesions by using an Fe(II)- and αKG-dependent oxidative demethylation mechanism. The structure of the active site features the facial triad of protein ligands consisting of the side chains of two histidine residues and one aspartate residue that is common to many non-heme Fe(II) oxygenases. X-ray absorption spectroscopy (XAS) of metallated (Fe and Ni) samples of ABH2 was used to investigate the mechanism of ABH2 and its inhibition by Ni(II) ions. The data are consistent with a sequential mechanism that features a five-coordinate metal center in the presence and absence of the α-ketoglutarate cofactor. This aspect is not altered in the Ni(II)-substituted enzyme, and both metals are shown to bind the cofactor. When the substrate is bound to the native Fe(II) complex with α-ketoglutarate bound, a five-coordinate Fe(II) center is retained that features an open coordination position for O(2) binding. However, in the case of the Ni(II)-substituted enzyme, the complex that forms in the presence of the cofactor and substrate is six-coordinate and, therefore, features no open coordination site for oxygen activation at the metal.     

Novel structural variation of silver(I)-pyridine complexes in nitromethane as studied by X-ray absorption spectroscopy

The XAFS spectra were measured at around the Ag K-edge of the Ag(I) ion in nitromethane (NM) with a variety of concentrations of pyridine (PY). In NM without PY, the Ag(I) ion is tetrahedrally solvated by four NM molecules similar to those in most solvents. The Ag-O bond length in NM solvent is longer than that in aqueous solution, indicating the low donating ability of NM. The mono-, bis-, tris-, and tetrakis-pyridine complexes are formed in NM by the addition of PY. The EXAFS analyses reveal that the structure of the formed PY complex in NM is linear for Ag(py)(nm)⁺, linear for , triangular for , and tetrahedral for . The longer Ag-O bond length for Ag(py)(nm)⁺ than that for and the release of bound NM molecules at the formation of Ag(py)(nm)⁺ are interpreted to be due to the strong σ donating property of PY. The Ag-N bond length (220 pm) for is intermediate between 216 pm for and 228 pm for . The formation equilibria of and are analyzed on the basis of the changeover of EXAFS spectra as a function of the total concentrations of Ag⁺ and PY in NM.     

Cavities and packing defects in the structural dynamics of myoglobin

Small globular proteins contain internal cavities and packing defects that reduce thermodynamic stability but seem to play a role in controlling function by defining pathways for the diffusion of the ligand/substrate to the active site. In the case of myoglobin (Mb), a prototype for structure–function relationship studies, the photosensitivity of the adduct of the reduced protein with CO, O₂ and NO allows events related to the migration of the ligand through the matrix to be followed. The crystal structures of intermediate states of wild-type (wt) and mutant Mbs show the photolysed CO to be located either in the distal heme pocket (primary docking site) or in one of two alternative cavities (secondary docking sites) corresponding to packing defects accessible to an atom of xenon. These results convey the general picture that pre-existing internal cavities are involved in controlling the dynamics and reactivity of the reactions of Mb with O₂ and other ligands, including NO.     

Analysis of the interaction of gallic acid and myoglobin by UV-vis absorption spectroscopy

UV-vis absorption spectroscopy has been used to analyze the interaction of myoglobin (Мb) and gallic acid (GA). The binding constants (4.38 × 10⁴ M^–1 at 298.15 K and 0.42 × 10⁴ М^–1 at 308.15K), the number of binding sites (h = 1.0), and the thermodynamic parameters of binding (ΔH, ΔS, and ΔG) have been determined. Hydrogen bonds have been shown to play a major role in the stabilization of the GA–Мb complexes. GA binding led to slight changes in the electronic state of the heme ring of the protein.     

Intermediate states in ligand photodissociation of carboxymyoglobin studied by dispersive X-ray absorption

The ligand photodissociation of sperm whale carboxymyoglobin (MbCO) at low temperature (15 K-100 K) under extended illumination has been studied by X-ray Absorption Near Edge Structure (XANES) spectroscopy using the dispersive technique. XANES simulations through the multiple scattering (MS) approach allow one to interpret the spectroscopic data in structural terms, and to investigate the Fe site structure configurations of the states that follow the CO photodissociation as a function of temperature. The Fe site in the photoproduct is unbound, with an overall structure similar to the deoxy-form (Mb) of the protein. The Fe site structure changes from T < 30 K (Mb^*) to T>50 K (Mb^**), revealing the existence of a slower unbound state Mb^**. A model is proposed which includes the faster state (Mb^*) as a planar porphyrin ring with a displacement of Fe from the heme plane of less than 0.3 Å, and the slower state (Mb**) with a domed heme. Correspondence to: S. Della Longa     

Protein conformation change of myoglobin upon ligand binding probed by ultraviolet resonance Raman spectroscopy

Conformational change of myoglobin (Mb) accompanied by binding of a ligand was investigated with 244 nm excited ultraviolet resonance Raman Spectroscopy (UVRR). The UVRR spectra of native sperm whale (sw) and horse (h) Mbs and W7F and W14F swMb mutants for the deoxy and CO-bound states enabled us to reveal the UVRR spectra of Trp7, Trp14, and Tyr151 residues, separately. The difference spectra between the deoxy and CO-bound states reflected the environmental or structural changes of Trp and Tyr residues upon CO binding. The W3 band of Trp7 near the N-terminus exhibited a change upon CO binding, while Trp14 did not. Tyr151 in the C-terminus also exhibited a definite change upon CO binding, but Tyr103 and Tyr146 did not. The spectral change of Tyr residues was characterized through solvent effects of a model compound. The corresponding spectral differences between CO- and n-butyl isocyanide-bound forms were much smaller than those between the deoxy and CO-bound forms, suggesting that the conformation change in the C- and N-terminal regions is induced by the proximal side of the heme through the movement of iron. Although the swinging up of His64 upon binding of a bulky ligand is noted by X-ray crystallographic analysis, UVRR spectra of His for the n-butyl isocyanide-bound form did not detect the exposure of His64 to solvent.     

X-ray absorption spectroscopy of Pyrococcus furiosus rubredoxin

X-ray absorption spectroscopy has been used to probe the frozen solution structure of the metal site in Pyrococcus furiosus rubredoxin in the native, iron-containing protein and in zinc- and mercury-substituted proteins. For all samples studied, the spectra have been interpreted in terms of a single shell of coordinated sulfur, with approximately tetrahedral coordination. For the native protein we obtain Fe-S bond-lengths of 2.29 and 2.33?Å for oxidized and reduced proteins, respectively. These values are in excellent agreement with those previously obtained from X-ray crystallography. The metal-substituted rubredoxins possess metal-sulfur bond lengths of 2.34 and 2.54?Å for the zinc- and mercury-substituted proteins, respectively.