The distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of two distal histidine tautomers.

Four independent 90 ps molecular dynamics simulations of sperm-whale wild-type carbonmonoxy myoglobin (MbCO) have been calculated using a new AMBER force field for the haem prosthetic group. Two trajectories have the distal 64N delta nitrogen protonated, and two have the 64N epsilon nitrogen protonated; all water molecules within 16 A of the carbonyl O are included. In three trajectories, the distal residue remains part of the haem pocket, with the protonated distal nitrogen pointing into the active site. This is in contrast with the neutron diffraction crystal structure, but is consistent with the solution phase CO stretching frequencies (upsilon CO) of MbCO and various of its mutants. There are significant differences in the "closed" pocket structures found for each tautomer: the 64N epsilon H trajectories both show stable distal-CO interactions, whereas the 64N delta H tautomer) has a weaker interaction resulting in a more mobile distal side chain. One trajectory (a 64N delta H tautomer) has the distal histidine moving out into the "solvent", leaving the pocket in an "open" structure, with a large unhindered entrance to the active site. These trajectories suggest that the three upsilon CO frequencies observed for wild-type MbCO in solution, rather than representing significantly different Fe-C-O geometries as such, arise from three different haem pocket structures, each with different electric fields at the ligand. Each pocket structure corresponds to a different distal histidine conformer: the A3 band to the 64N epsilon H tautomer, the A1,2 band to the 64N delta H tautomer, and the A0 band to the absence of any significant interaction with the distal side chain.     

Distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of three distal mutants.

Six 90-ps molecular dynamics trajectories, two for each of three distal mutants of sperm whale carbonmonoxy myoglobin, are reported; solvent waters within 16 A of the active site have been included. In both His64GIn trajectories, the distal side chain remains part of the heme pocket, forming a "closed" conformation similar to that of the wild type 64N delta H tautomer. Despite a connectivity more closely resembling the N epsilon H histidine tautomer, close interactions with the carbonyl ligand similar to those observed for the wild type 64N epsilon H tautomer are prevented in this mutant by repulsive interactions between the carbonyl O and the 64O epsilon. The aliphatic distal side chain of the His64Leu mutant shows little interaction with the carbonyl ligand in either His64Leu trajectory. Solvent water molecules move into and out of the active site in the His64Gly mutant trajectories; during all the other carbonmonoxy myoglobin trajectories, including the wild type distal tautomers considered in an earlier work, solvent molecules rarely encroach closer than 6 A of the active site. These results are consistent with a recent structural interpretation of the wild type infrared spectrum, and the current reinterpretation that the distal-ligand interaction in carbonmonoxy myoglobin is largely electrostatic, not steric, in nature.     

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 His_ɛ⁶⁴ 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.     

Rearrangement of the distal pocket accompanying E7 His----Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket

Two-dimensional 1H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO2). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C zeta H proton of the Phe CD4 was found to move toward the iron of the heme by approximately 4 A relative to the position of sperm whale MbCO, requiring minimally a 3-A movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His----Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.     

Low-barrier hydrogen bond hypothesis in the catalytic triad residue of serine proteases: correlation between structural rearrangement and chemical shifts in the acylation process

To elucidate the catalytic advantage of the low-barrier hydrogen bond (LBHB), we analyze the hydrogen bonding network of the catalytic triad (His57-Asp102-Ser195) of serine protease trypsin, one of the best examples of the LBHB reaction mechanism. Especially, we focus on the correlation between the change of the chemical shifts and the structural rearrangement of the active site in the acylation process. To clarify LBHB, we evaluate the two complementary properties. First, we calculate the NMR chemical shifts of the imidazole ring of His57 by the gauge-including atomic orbital (GIAO) approach within the ab initio QM/MM framework. Second, the free energy profile of the proton transfer from His57 to Asp102 in the tetrahedral intermediate is obtained by ab initio QM/MM calculations combined with molecular dynamics free energy perturbation (MD-FEP) simulations. The present analyses reveal that the calculated shifts reasonably reproduce the observed values for (1)H chemical shift of H(epsilon)(1) and H(delta)(1) in His57. The (15)N and (13)C chemical shifts are also consistent with the experiments. It is also shown that the proton between His57 and Asp102 is localized at the His57 side. This largely downfield chemical shift is originated from the strong electrostatic interaction, not a covalent-like bonding character between His57 and Asp102. Also, it is proved that a slight downfield character of H(epsilon)(1) is originated from a electrostatic interaction between His57 and the backbone carbonyl group of Val213 and Ser214. These downfield chemical shifts are observed only when the tetrahedral intermediate is formed in the acylation process.     

Structural dynamics of distal histidine replaced mutants of myoglobin accompanied with the photodissociation reaction of the ligand

Protein dynamics observed by the transient grating (TG) method are studied for some site-directed mutants at the distal histidine of myoglobin (H64L, H64Q, H64V). The time profiles of the TG signals are very sensitive to the amino acid residue of the 64 position. It was found that the sensitivity is mostly caused by the different rates of the ligand escape from the protein to solvent and the magnitude of the molecular volume change. Several molecular origins of the volume difference between MbCO and Mb, such as the electrostatic interaction in the distal pocket, movement of helices, and distal water, are proposed. Interestingly, the volume difference between the CO-trapped Mb inside the protein interior and Mb is similar to that of the partial molar volume of CO in organic solvent. The effect of mutation on the nature of the CO trapped site is discussed.     

Kinetic, structural, and spectroscopic identification of geminate states of myoglobin: a ligand binding site on the reaction pathway

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.     

Crystal structures of ferrous horse heart myoglobin complexed with nitric oxide and nitrosoethane

The interactions of nitric oxide (NO) and organic nitroso compounds with heme proteins are biologically important, and adduct formation between NO-containing compounds and myoglobin (Mb) have served as prototypical systems for studies of these interactions. We have prepared crystals of horse heart (hh) MbNO from nitrosylation of aqua-metMb crystals, and we have determined the crystal structure of hh MbNO at a resolution of 1.9 A. The Fe-N-O angle of 147 degrees in hh MbNO is larger than the corresponding 112 degrees angle previously determined from the crystal structure of sperm whale MbNO (Brucker et al., Proteins 1998;30:352-356) but is similar to the 150 degrees angle determined from a MS XAFS study of a frozen solution of hh MbNO (Rich et al., J Am Chem Soc 1998;120:10827-10836). The Fe-N(O) bond length of 2.0 A (this work) is longer than the 1.75 A distance determined from the XAFS study and suggests distal pocket influences on FeNO geometry. The nitrosyl N atom is located 3.0 A from the imidazole N(epsilon) atom of the distal His64 residue, suggesting electrostatic stabilization of the FeNO moiety by His64. The crystal structure of the nitrosoethane adduct of ferrous hh Mb was determined at a resolution of 1.7 A. The nitroso O atom of the EtNO ligand is located 2.7 A from the imidazole N(epsilon) atom of His64, suggesting a hydrogen bond interaction between these groups. To the best of our knowledge, the crystal structure of hh Mb(EtNO) is the first such determination of a nitrosoalkane adduct of a heme protein.     

Theoretical investigation of infrared spectra and pocket dynamics of photodissociated carbonmonoxy myoglobin

Molecular dynamics simulations of the photodissociated state of carbonmonoxy myoglobin (MbCO) are presented using a fluctuating charge model for CO. A new three-point charge model is fitted to high-level ab initio calculations of the dipole and quadrupole moment functions taken from the literature. The infrared spectrum of the CO molecule in the heme pocket is calculated using the dipole moment time autocorrelation function and shows good agreement with experiment. In particular, the new model reproduces the experimentally observed splitting of the CO absorption spectrum. The splitting of 3-7 cm(-1) (compared to the experimental value of 10 cm(-1)) can be directly attributed to the two possible orientations of CO within the docking site at the edge of the distal heme pocket (the B states), as previously suggested on the basis of experimental femtosecond time-resolved infrared studies. Further information on the time evolution of the position and orientation of the CO molecule is obtained and analyzed. The calculated difference in the free energy between the two possible orientations (Fe...CO and Fe...OC) is 0.3 kcal mol(-1) and agrees well with the experimentally estimated value of 0.29 kcal mol(-1). A comparison of the new fluctuating charge model with an established fixed charge model reveals some differences that may be critical for the correct prediction of the infrared spectrum and energy barriers. The photodissociation of CO from the myoglobin mutant L29F using the new model shows rapid escape of CO from the distal heme pocket, in good agreement with recent experimental data. The effect of the protein environment on the multipole moments of the CO ligand is investigated and taken into account in a refined model. Molecular dynamics simulations with this refined model are in agreement with the calculations based on the gas-phase model. However, it is demonstrated that even small changes in the electrostatics of CO alter the details of the dynamics.     

A study of vibrational relaxation of B-state carbon monoxide in the heme pocket of photolyzed carboxymyoglobin.

The vibrational energy relaxation of dissociated carbon monoxide in the heme pocket of sperm whale myoglobin has been studied using equilibrium molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the delta- and epsilon-tautomers of the distal histidine, His64. Vibrational population relaxation times were estimated using the Landau-Teller model. For carbon monoxide (CO) in the myoglobin epsilon-tautomer, for a frequency of omega0 = 2131 cm-1 corresponding to the B1 state, T1epsilon(B1) = 640 +/- 185 ps, and for a frequency of omega0 = 2119 cm-1 corresponding to the B2 state, T1epsilon(B2) = 590 +/- 175 ps. Although the CO relaxation rates in both the epsilon- and delta-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the delta-tautomer. For CO in the myoglobin delta-tautomer, it was found that the relaxation times were identical within error for the two CO substate frequencies, T1delta(B1) = 335 +/- 115 ps and T1delta(B2) = 330 +/- 145 ps. These simulation results are in reasonable agreement with experimental results of Anfinrud and coworkers (unpublished results). Normal mode calculations were used to identify the dominant coupling between the protein and CO molecules. The calculations suggest that the residues of the myoglobin pocket, acting as a first solvation shell to the CO molecule, contribute the primary "doorway" modes in the vibrational relaxation of the oscillator.     

Crystal structures of myoglobin-ligand complexes at near-atomic resolution.

We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 A. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 A root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 +/- 3 degrees with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO(2) seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.     

Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers

X-ray structures of carbonmonoxymyoglobin (MbCO) are available for different pH values. We used conventional electrostatic continuum methods to calculate the titration behavior of MbCO in the pH range from 3 to 7. For our calculations, we considered five different x-ray structures determined at pH values of 4, 5, and 6. We developed a Monte Carlo method to sample protonation states and conformations at the same time so that we could calculate the population of the considered MbCO structures at different pH values and the titration behavior of MbCO for an ensemble of conformers. To increase the sampling efficiency, we introduced parallel tempering in our Monte Carlo method. The calculated population probabilities show, as expected, that the x-ray structures determined at pH 4 are most populated at low pH, whereas the x-ray structure determined at pH 6 is most populated at high pH, and the population of the x-ray structures determined at pH 5 possesses a maximum at intermediate pH. The calculated titration behavior is in better agreement with experimental results compared to calculations using only a single conformation. The most striking feature of pH-dependent conformational changes in MbCO-the rotation of His-64 out of the CO binding pocket-is reproduced by our calculations and is correlated with a protonation of His-64, as proposed earlier.     

Residues in the distal heme pocket of neuroglobin. Implications for the multiple ligand binding steps

Amino acid residues in the ligand binding pocket of human neuroglobin have been identified by site-directed mutagenesis and their properties investigated by resonance Raman and flash photolysis methods. Wild-type neuroglobin has been shown to have six-coordinate heme in both ferric and ferrous states. Substitution of His96 by alanine leads to complete loss of heme, indicating that His96 is the proximal ligand. The resonance Raman spectra of M69L and K67T mutants were similar to those of wild-type (WT) neuroglobin in both ferric and ferrous states. By contrast, H64V was six-coordinate high-spin and five-coordinate high-spin in the ferric and ferrous states, respectively, at acidic pH. The spectra were pH-dependent and six-coordinate with the low-spin component dominating at alkaline pH. In a double mutant H64V/K67T, the high-spin component alone was detected in the both ferric and the ferrous states. This implies that His64 is the endogenous ligand and that Lys67 is situated nearby in the distal pocket. In the ferrous H64V and H64V/K67T mutants, the nu(Fe-His) stretching frequency appears at 221 cm(-1), which is similar to that of deoxymyoglobin. In the ferrous CO-bound state, the nu(Fe-CO) stretching frequency was detected at 521 and 494 cm(-1) in WT, M69L, and K67T, while only the 494 cm(-1) component was detected in the H64V and H64V/K67T mutants. Thus, the 521 cm(-1) component is attributed to the presence of polar His64. The CO binding kinetics were biphasic for WT, H64V, and K67T and monophasic for H64V/K67T. Thus, His64 and Lys67 comprise a unique distal heme pocket in neuroglobin.     

Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.     

Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release

HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.     

Computational and experimental studies on the catalytic mechanism of biliverdin-IXbeta reductase

BVR-B (biliverdin-IXbeta reductase) also known as FR (flavin reductase) is a promiscuous enzyme catalysing the pyridine-nucleotide-dependent reduction of a variety of flavins, biliverdins, PQQ (pyrroloquinoline quinone) and ferric ion. Mechanistically it is a good model for BVR-A (biliverdin-IXalpha reductase), a potential pharmacological target for neonatal jaundice and also a potential target for adjunct therapy to maintain protective levels of biliverdin-IXalpha during organ transplantation. In a commentary on the structure of BVR-B it was noted that one outstanding issue remained: whether the mechanism was a concerted hydride transfer followed by protonation of a pyrrolic anion or protonation of the pyrrole followed by hydride transfer. In the present study we have attempted to address this question using QM/MM (quantum mechanics/molecular mechanics) calculations. QM/MM potential energy surfaces show that the lowest energy pathway proceeds with a positively charged pyrrole intermediate via two transition states. These initial calculations were performed with His(153) as the source of the proton. However site-directed mutagenesis studies with both the H153A and the H153N mutant reveal that His(153) is not required for catalytic activity. We have repeated the calculation with a solvent hydroxonium donor and obtain a similar energy landscape indicating that protonation of the pyrrole is the most likely first step followed by hydride transfer and that the required proton may come from bulk solvent. The implications of the present study for the design of inhibitors of BVR-A are discussed.     

X-ray structure and refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 A resolution

The structure of carbon-monoxy (Fe II) myoglobin at 260 K has been solved at a resolution of 1.5 A by X-ray diffraction and a model refined against the X-ray data by restrained least-squares. The CO ligand is disordered and distorted from the linear conformation seen in model compounds. At least two conformations, with Fe--C--O angles of 140 degrees and 120 degrees, are required to model the system. The heme pocket is significantly larger than in deoxy-myoglobin because the distal residues have relaxed around the ligand; the largest displacement occurs for the distal histidine side-chain, which moves more than 1.4 A on ligand binding. The side-chain of Arg45 (CD3) is disordered and apparently exists in two equally populated conformations. One of these does not block the motion of the distal histidine out of the binding pocket, suggesting a mechanism for ligand entry. The heme group is planar (root-mean-square deviation from planarity is 0.08 A) with no doming of the pyrrole groups. The Fe--N epsilon 2 (His93) bond length is 2.2 A and the Fe--C bond length in the CO complex is 1.9 A. The iron is the least-squares plane of the heme, and this leads to the proximal histidine moving by 0.4 A relative to its position in deoxy-myoglobin. This shift correlates with a global structural change, with the proximal part of the molecule translated towards the heme plane.     

Structural and kinetic analysis of proton shuttle residues in the active site of human carbonic anhydrase III

We report the X-ray crystal structures and rate constants for proton transfer in site-specific mutants of human carbonic anhydrase III (HCA III) that place a histidine residue in the active-site cavity: K64H, R67H, and K64H-R67N HCA III. Prior evidence from the exchange of 18O between CO2 and water measured by mass spectrometry shows each mutant to have enhanced proton transfer in catalysis compared with wild-type HCA III. However, His64 in K64H and K64H-R67N HCA III have at most a capacity for proton transfer that is only 13% that of His64 in HCA II. This reduced rate in mutants of HCA III is associated with a constrained side-chain conformation of His64, which is oriented outward, away from the active-site zinc in the crystal structures. This conformation appears stabilized by a prominent pi stacking interaction of the imidazole ring of His64 with the indole ring of Trp5 in mutants of HCA III. This single orientation of His64 in K64H HCA III predominates also in a double mutant K64H-R67N HCA III, indicating that the positive charge of Arg67 does not influence the observed conformation of His64 in the crystal structure. Hence, the structures and catalytic activity of these mutants of HCA III containing His64 account only in small part for the lower activity of this isozyme compared with HCA II. His67 in R67H HCA III was also shown to be a proton shuttle residue, having a capacity for proton transfer that was approximately four times that of His64 in K64H HCA III. This is most likely due to its proximity and orientation inward towards the zinc-bound solvent. These results emphasize the significance of side chain orientation and range of available conformational states as characteristics of an efficient proton shuttle in carbonic anhydrase.     

The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined QM/MM simulations

Molecular dynamics simulations employing combined quantum mechanical and molecular mechanical (QM/MM) potentials have been carried out to investigate the reaction mechanism of the hydrolysis of paraoxon by phosphotriesterase (PTE). We used a dual-level QM/MM approach that synthesizes accurate results from high-level electronic structure calculations with computational efficiency of semiempirical QM/MM potentials for free energy simulations. In particular, the intrinsic (gas-phase) energies of the active site in the QM region are determined by using density functional theory (B3LYP) and second-order M?ller-Plesset perturbation theory (MP2) and the molecular dynamics free energy simulations are performed by using the mixed AM1:CHARMM potential. The simulation results suggest a revised mechanism for the phosphotriester hydrolysis mechanism by PTE. The reaction free energy profile is mirrored by structural motions of the binuclear metal center in the active site. The two zinc ions occupy a compact conformation with an average zinc-zinc distance of 3.5 +/- 0.1 A in the Michaelis complex, whereas it is elongated to 5.3 +/- 0.3 A at the transition state and product state. The substrate is loosely bound to the more exposed zinc ion (Znbeta2+) at an average distance of 3.8 A +/- 0.3 A. The P=O bond of the substrate paraoxon is activated by adopting a tight coordination to the Znbeta2+, releasing the coordinate to the bridging hydroxide ion and increasing its nucleophilicity. It was also found that a water molecule enters into the binding pocket of the loosely bound binuclear center, originally occupied by the nucleophilic hydroxide ion. We suggest that the proton of this water molecule is taken up by His254 at low pH or released to the solvent at high pH, resulting in a hydroxide ion that pulls the Znbeta2+ ion closer to form the compact configuration and restores the resting state of the enzyme.