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MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 μM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.

The gas-phase free energy differences between 20 phenols and their respective anions are estimated by CBS-QB3 method and the CPCM continuum solvation method is employed to calculate the free energy differences of solvation for the phenols and their anions. Absolute pKa calculations with solvated phase of optimized structures for the CPCM calculation yield standard deviations and root-mean-square errors of less than 0.4-pKa units, which is critical and of great importance.

Complete basis set and Gaussian-n models were combined with CPCM continuum solvation methods to gain accurate pKa calculations for caboxylic acids.

The GAUSSIAN 2, GAUSSIAN 3, complete basis set-QB3, and complete basis set-APNO methods have been used to calculate ΔH° and ΔG° values for ionic clusters of hydronium and hydroxide ions complexed with water. Results for the clusters H3O+(H2O)n and OH-(H2O)n, where n=1–4 are reported in this paper, and compared against experimental values contained in the National Institutes of Standards and Technology (NIST) database. Agreement with experiment is excellent for the three ab initio methods for formation of these clusters. The high accuracy of these methods makes them reliable for calculating energetics for the formation of ionic clusters containing water. In addition this allows them to serve as a valuable check on the accuracy of experimental data reported in the NIST database, and makes them useful tools for addressing unresolved issues in atmospheric chemistry. © 2005 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The I690C/G743C variant of a C-terminal fragment of the cobalamin-dependent methionine synthase (MetH) is prepared to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. The S-adenosylmethionine substrate requirement has provided MetH a means to suppress deleterious side reactions involving the transiently formed cob(I)alamin ([Co.sup.1+]Cbl) supernucleophile.

Fast protein liquid chromatography (FPLC) is used to separate two distinct species of the [Co.sup.2+]Cbl-bound H759G MetH mutant, which have differed in their ability to catalyze the reductive methylation of the cofactor. The formation of the four-coordinate cob(II)alamin dead-end species in the His759Gly variant has highlighted the importance of the His759 residue in governing the equilibria between the different conformations of MetH.

The nickel chelatase CfbA is the smallest member of the chelatase family, but the mechanism by which this enzyme inserts nickel into sirohydrochlorin is unknown. In order to gain mechanistic insight, metal binding, tetrapyrrole binding, and enzyme activity were characterized for a variety of substrates using several spectroscopic and computational approaches. Mass spectrometery and magnetic circular dichroism experiments revealed that CfbA binds an octahedral, high-spin metal substrate. UV/Vis absorption spectroscopy demonstrated that the enzyme binds a wide range of tetrapyrrole substrates and perturbs their electronic structures. Based upon activity assays, CfbA promotes insertion of cobalt and nickel into several tetrapyrroles, including cobalt insertion into protopophyrin IX. Finally, density functional theory models were developed which strongly suggest that observed spectral changes upon binding to the enzyme can be explained by tetrapyrrole ruffling, but not deprotonation or saddling. The observation of an octahedral, high-spin metal bound to CfbA leads to a generalization for all class II chelatases: these enzymes bind labile metal substrates and metal desolvation is not a rate-limiting step. The conclusion that CfbA ruffles its tetrapyrrole substrate reveals that the CfbA mechanism is different from that currently proposed for ferrochelatase, and identifies an intriguing correlation between metal substrate specificity and tetrapyrrole distortion mode in chelatases. [ABSTRACT FROM AUTHOR]

The cobalamin-dependent methionine synthase (MetH) from Escherichia co/i is a modular enzyme that catalyzes a methyl group transfer from methyltetrahydrofolate to homocysteine via a methylcob(lll)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every ∼2000 turnovers, the cobalamin cofactor is converted to the inactive cob(ll)alamin (Co2+Cbl) form, from which MeCbl has to be recovered for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co2+CbI species to cob(l)alamin (Co1+Cbl) by flavodoxin, a reaction that would appear to be endergonic on the basis of the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the 1690C/G743C variant of a C-terminal fragment of MetH (MetHCT) to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the 1690C/G743C1Y1 1 39F MetHCT triple mutant reveals that the strategy employed by MetH to activate Co2+Cbl for Co2+ - Co1+ reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co-OH2 bond of the cofactor, thereby causing a large stabilization of the Co 3d2-based "redox-active" molecular orbital. The lengthening of the Co-OH2 bond is mediated by the Yl 139 active-site residue and becomes much more dramatic when the S-adenosylme- thionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co1+Cbl "supernucleophile". [ABSTRACT FROM AUTHOR]

Determines the gas-phase free energy differences in phenols and its anions. Charge separation between the negatively charged oxygen and the ring system of phenoxide anions; Structures of the anions; Use of supramolecule-continuum approach in estimating the free energy of proton hydration.