Spectroscopic and computational characterization of the base-off forms of cob(II)alamin.
ABSTRACT: The one-electron-reduced form of vitamin B(12), cob(II)alamin (Co(2+)Cbl), is found in several essential human enzymes, including the cobalamin-dependent methionine synthase (MetH). In this work, experimentally validated electronic structure descriptions for two "base-off" Co(2+)Cbl species have been generated using a combined spectroscopic and computational approach, so as to obtain definitive clues as to how these and related enzymes catalyze the thermodynamically challenging reduction of Co(2+)Cbl to cob(I)alamin (Co(1+)Cbl). Specifically, electron paramagnetic resonance (EPR), electronic absorption (Abs), and magnetic circular dichroism (MCD) spectroscopic techniques have been employed as complementary tools to characterize the two distinct forms of base-off Co(2+)Cbl that can be trapped in the H759G variant of MetH, one containing a five-coordinate and the other containing a four-coordinate, square-planar Co(2+) center. Accurate spin Hamiltonian parameters for these low-spin Co(2+) centers have been determined by collecting EPR data using both X- and Q-band microwave frequencies, and Abs and MCD spectroscopic techniques have been employed to probe the corrin-centered pi --> pi* and Co-based d --> d excitations, respectively. By using these spectroscopic data to evaluate electronic structure calculations, we found that density functional theory provides a reasonable electronic structure description for the five-coordinate form of base-off Co(2+)Cbl. However, it was necessary to resort to a multireference ab initio treatment to generate a more realistic description of the electronic structure of the four-coordinate form. Consistent with this finding, our computational data indicate that, in the five-coordinate Co(2+)Cbl species, the unpaired spin density is primarily localized in the Co 3d(z(2))-based molecular orbital, as expected, whereas in the four-coordinate form, extensive Co 3d orbital mixing, configuration interaction, and spin-orbit coupling cause the unpaired electron to delocalize over several Co 3d orbitals. These results provide important clues to the mechanism of enzymatic Co(2+)Cbl --> Co(1+)Cbl reduction.
Project description:EutT from Salmonella enterica is a member of a class of enzymes termed ATP:Co(I)rrinoid adenosyltransferases (ACATs), implicated in the biosynthesis of adenosylcobalamin (AdoCbl). In the presence of cosubstrate ATP, ACATs raise the Co(II)/Co(I) reduction potential of their cob(II)alamin [Co(II)Cbl] substrate by >250 mV via the formation of a unique four-coordinate (4c) Co(II)Cbl species, thereby facilitating the formation of a "supernucleophilic" cob(I)alamin intermediate required for the formation of the AdoCbl product. Previous kinetic studies of EutT revealed the importance of a HX11CCX2C(83) motif for catalytic activity and have led to the proposal that residues in this motif serve as the binding site for a divalent transition metal cofactor [e.g., Fe(II) or Zn(II)]. This motif is absent in other ACAT families, suggesting that EutT employs a distinct mechanism for AdoCbl formation. To assess how metal ion binding to the HX11CCX2C(83) motif affects the relative yield of 4c Co(II)Cbl generated in the EutT active site, we have characterized several enzyme variants by using electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies. Our results indicate that Fe(II) or Zn(II) binding to the HX11CCX2C(83) motif of EutT is required for promoting the formation of 4c Co(II)Cbl. Intriguingly, our spectroscopic data also reveal the presence of an equilibrium between five-coordinate "base-on" and "base-off" Co(II)Cbl species bound to the EutT active site at low ATP concentrations, which shifts in favor of "base-off" Co(II)Cbl in the presence of excess ATP, suggesting that the base-off species serves as a precursor to 4c Co(II)Cbl.
Project description:The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HX11CCX2C(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin. Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HX11CCX2C(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HX11CCX2C(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutTWT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutTWT/Co relative to that of the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data for EutTWT/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution of either His67, His75, Cys79, Cys80, or Cys83 indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.
Project description:The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every approximately 2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co(2+)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 Co(2+)Cbl species to cob(I)alamin (Co(1+)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 I690C/G743C variant of a C-terminal fragment of MetH (MetH(CT)) 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 I690C/G743C/Y1139F MetH(CT) triple mutant reveals that the strategy employed by MetH to activate Co(2+)Cbl for Co(2+) --> Co(1+) 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-OH(2) bond of the cofactor, thereby causing a large stabilization of the Co 3d(z(2))-based "redox-active" molecular orbital. The lengthening of the Co-OH(2) bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co(1+)Cbl "supernucleophile".
Project description:The EutT enzyme from Listeria monocytogenes ( LmEutT) is a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes that catalyze the biosynthesis of adenosylcobalamin (AdoCbl) from exogenous Co(II)rrinoids and ATP. Apart from EutT-type ACATs, two evolutionary unrelated types of ACATs have been identified, termed PduO and CobA. Although the three types of ACATs are nonhomologous, they all generate a four-coordinate cob(II)alamin (4C Co(II)Cbl) species to facilitate the formation of a supernucleophilic Co(I)Cbl intermediate capable of attacking the 5'-carbon of cosubstrate ATP. Previous spectroscopic studies of the EutT ACAT from Salmonella enterica ( SeEutT) revealed that this enzyme requires a divalent metal cofactor for the conversion of 5C Co(II)Cbl to a 4C species. Interestingly, LmEutT does not require a divalent metal cofactor for catalytic activity, which exemplifies an interesting phylogenetic divergence among the EutT enzymes. To explore if this disparity in the metal cofactor requirement among EutT enzymes correlates with differences in substrate specificity or the mechanism of Co(II)Cbl reduction, we employed various spectroscopic techniques to probe the interaction of Co(II)Cbl and cob(II)inamide (Co(II)Cbi+) with LmEutT in the absence and presence of cosubstrate ATP. Our data indicate that LmEutT displays a similar substrate specificity as SeEutT and can bind both Co(II)Cbl and Co(II)Cbi+ when complexed with MgATP, though it exclusively converts Co(II)Cbl to a 4C species. Notably, LmEutT is the most effective ACAT studied to date in generating the catalytically relevant 4C Co(II)Cbl species, achieving a >98% 5C ? 4C conversion yield on the addition of just over one mol equiv of cosubstrate MgATP.
Project description:ATP:Corrinoid adenosyltransferases (ACAs) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the alpha face of the corrin ring consists of bulky hydrophobic residues. To understand how these residues promote the generation of the four-coordinate cob(II)alamin, variants of the human-type ACA enzyme from L. reuteri (LrPduO) were kinetically and structurally characterized. These studies revealed that residue Phe112 is critical in the displacement of 5,6-dimethylbenzimidazole (DMB) from its coordination bond with the Co ion of the ring, resulting in the formation of the four-coordinate species. An F112A substitution resulted in a 80% drop in the catalytic efficiency of the enzyme. The explanation for this loss of activity was obtained from the crystal structure of the mutant protein, which showed cob(II)alamin bound in the active site with DMB coordinated to the cobalt ion. The crystal structure of an LrPduO(F112H) variant showed a DMB-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency was 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of LrPduO(F112H) suggests that the F112H substitution negatively impacts product release. Substitutions of other hydrophobic residues in the Cbl binding pocket did not result in significant defects in catalytic efficiency in vitro; however, none of the variant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.
Project description:Cobalamin-dependent methionine synthase (MetH) of Escherichia coli is a 136 kDa, modular enzyme that undergoes large conformational changes as it uses a cobalamin cofactor as a donor or acceptor in three separate methyl transfer reactions. At different points during the reaction cycle, the coordination to the cobalt of the cobalamin changes; most notably, the imidazole side chain of His759 that coordinates to the cobalamin in the "His-on" state can dissociate to produce a "His-off" state. Here, two distinct species of the cob(II)alamin-bound His759Gly variant have been identified and separated. Limited proteolysis with trypsin was employed to demonstrate that the two species differ in protein conformation. Magnetic circular dichroism and electron paramagnetic resonance spectroscopies were used to show that the two species also differ with respect to the axial coordination to the central cobalt ion of the cobalamin cofactor. One form appears to be in a conformation poised for reductive methylation with adenosylmethionine; this form was readily reduced to cob(I)alamin and subsequently methylated [albeit yielding a unique, five-coordinate methylcob(III)alamin species]. Our spectroscopic data revealed that this form contains a five-coordinate cob(II)alamin species, with a water molecule as an axial ligand to the cobalt. The other form appears to be in a catalytic conformation and could not be reduced to cob(I)alamin under any of the conditions tested, which precluded conversion to the methylcob(III)alamin state. This form was found to possess an effectively four-coordinate cob(II)alamin species that has neither water nor histidine coordinated to the cobalt center. The formation of this four-coordinate cob(II)alamin "dead-end" species in the His759Gly variant illustrates the importance of the His759 residue in governing the equilibria between the different conformations of MetH.
Project description:Our mechanistic understanding of the conversion of vitamin B(12) into coenzyme B(12) (a.k.a. adenosylcobalamin, AdoCbl) has been substantially advanced in recent years. Insights into the multiple roles played by ATP:cob(I)alamin adenosyltransferase (ACA) enzymes have emerged through the crystallographic, spectroscopic, biochemical, and mutational analyses of wild-type and variant proteins. ACA enzymes circumvent the thermodynamic barrier posed by the very low redox potential associated with the reduction of cob(II)alamin to cob(I)alamin by generating a unique four-coordinate cob(II)alamin intermediate that is readily converted to cob(I)alamin by physiological reductants. ACA enzymes not only synthesize AdoCbl but also they deliver it to the enzymes that use it, and in some cases, enzymes in which its function is needed to maintain the fidelity of the AdoCbl delivery process have been identified. Advances in our understanding of ACA enzyme function have provided valuable insights into the role of specific residues, and into why substitutions of these residues have profound negative effects on human health. From an applied science standpoint, a better understanding of the adenosylation reaction may lead to more efficient ways of synthesizing AdoCbl.
Project description:ATP:cob(I)alamin adenosyltransferases (ACAs) catalyze the transfer of the 5'-deoxyadenosyl moiety from ATP to the upper axial ligand position of cobalamin in the synthesis of coenzyme B 12. For the ACA-catalyzed reaction to proceed, cob(II)alamin must be reduced to cob(I)alamin in the enzyme active site. This reduction is facilitated through the generation of a four-coordinate cob(II)alamin intermediate on the enzyme. We have determined the high-resolution crystal structure of a human-type ACA from Lactobacillus reuteri with a four-coordinate cob(II)alamin bound in the enzyme active site and with the product, adenosylcobalamin, partially occupied in the active site. The assembled structures represent snapshots of the steps in the ACA-catalyzed formation of the cobalt-carbon bond of coenzyme B 12. The structures define the corrinoid binding site and provide visual evidence for a base-off, four-coordinate cob(II)alamin intermediate. The complete structural description of ACA-mediated catalysis reveals the molecular features of four-coordinate cob(II)alamin stabilization and provides additional insights into the molecular basis for dysfunction in human patients suffering from methylmalonic aciduria.
Project description:The reaction of adenosylcobalamin-dependent dioldehydrase with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X-band EPR spectroscopy was performed in the presence of D(2)O and compared to the spectrum in H(2)O. Results indicate that the unpaired electron of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the (205,203)Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.
Project description:We studied the kinetics of reactions of cob(I)alamin and cob(I)inamide with thiosulfate, sulfite, and dithionite by UV-Visible (UV-Vis) and stopped-flow spectroscopy. We found that the two Co(I) species were oxidized by these sulfur-containing compounds to Co(II) forms: oxidation by excess thiosulfate leads to penta-coordinate complexes and oxidation by excess sulfite or dithionite leads to hexa-coordinate Co(II)-SO2(-) complexes. The net scheme involves transfer of three electrons in the case of oxidation by thiosulfate and one electron for oxidation by sulfite and dithionite. On the basis of kinetic data, the nature of the reactive oxidants was suggested, i.e., HS2O3(-) (for oxidation by thiosulfate), S2O5(2-), HSO3(-), and aquated SO2 (for oxidation by sulfite), and S2O4(2-) and SO2(-) (for oxidation by dithionite). No difference was observed in kinetics with cob(i)alamin or cob(i)inamide as reductants.