The reactivity of individual solvent-coupled protein configurations is used to track and resolve the progress coordinate for the core reaction sequence of substrate radical rearrangement and hydrogen atom transfer in the ethanolamine ammonia-lyase (EAL) enzyme from Salmonella enterica. The first-order decay of the substrate radical intermediate is the monitored reaction. Heterogeneous confinement from sucrose hydrates in the mesophase solvent surrounding the cryotrapped protein introduces distributed kinetics in the non-native decay of the substrate radical pair capture substate, which arise from an ensemble of configurational microstates. Reaction rates increase by >103-fold across the distribution to approach that for the native enabled substate for radical rearrangement, which reacts with monotonic kinetics. The native progress coordinate thus involves a collapse of the configuration space to generate optimized reactivity. Reactivity tracking reveals fundamental features of solvent-protein-reaction configurational coupling and leads to a model that refines the ensemble paradigm of enzyme catalysis for strongly adiabatic chemical steps.

CblC is a chaperone that catalyzes removal of the β-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the β-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.

The CblC and CblD chaperones are involved in early steps in the cobalamin trafficking pathway. Cobalamin derivatives entering the cytoplasm are converted by CblC to a common cob(II)alamin intermediate via glutathione-dependent alkyltransferase or reductive elimination activities. Cob(II)alamin is subsequently converted to one of two biologically active alkylcobalamins by downstream chaperones. The function of CblD has been elusive although it is known to form a complex with CblC under certain conditions. Here, we report that CblD provides a sulfur ligand to cob(II)alamin bound to CblC, forming an interprotein coordination complex that rapidly oxidizes to thiolato-cob(III)alamin. Cysteine scanning mutagenesis and EPR spectroscopy identified Cys-261 on CblD as the sulfur donor. The unusual interprotein Co-S bond was characterized by X-ray absorption spectroscopy and visualized in the crystal structure of the human CblD thiolato-cob(III)alamin complex. Our study provides insights into how cobalamin coordination chemistry could be utilized for cofactor translocation in the trafficking pathway.

IcmF is a 5′-deoxyadenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the carbon skeleton rearrangement of isobutyryl-CoA to butyryl-CoA. It is a bifunctional protein resulting from the fusion of a G-protein chaperone with GTPase activity and the cofactor- and substrate-binding mutase domains with isomerase activity. IcmF is prone to inactivation during catalytic turnover, thus setting up its dependence on a cofactor repair system. Herein, we demonstrate that the GTPase activity of IcmF powers the ejection of the inactive cob(II)alamin cofactor and requires the presence of an acceptor protein, adenosyltransferase, for receiving it. Adenosyltransferase in turn converts cob(II)alamin to AdoCbl in the presence of ATP and a reductant. The repaired cofactor is then reloaded onto IcmF in a GTPase-gated step. The mechanistic details of cofactor loading and offloading from the AdoCbl-dependent IcmF are distinct from those of the better characterized and homologous methylmalonyl-CoA mutase/G-protein chaperone system.

The amyloid-β (Aβ) protein forms fibrils and higher-order plaque aggegrates in Alzheimer's disease (AD) brain. The copper ion, Cu2+, is found at high concentrations in plaques, but its role in AD etiology is unclear. We use high-resolution pulsed-electron paramagnetic resonance (EPR) spectroscopy to characterize the coordination structure of Cu2+ in the fibrillar form of full-length Aβ(1-40). The results reveal a bis-cis-histidine (His) equatorial Cu2+ coordination geometry, and participation of all three N-terminal His residues in Cu2+ binding. A model is proposed, in which Cu2+–His6/His13 and Cu2+–His6/His14 sites alternate along the fibril axis, on opposite sides of the β-sheet fibril structure. The local intra-β-strand coordination structure is not conducive to Cu2+/Cu1+ redox-linked coordination changes, and the global arrangement of Cu sites precludes facile multi-electron and bridged-metal site reactivity. This indicates that the fibrillar form of Aβ suppresses Cu redox cycling and reactive oxygen species (ROS) production. The insulator configuration suggests application of Cu2+-Aβ fibrils as an amyloid architecture for switchable electron charge/spin coupling and redox reactivity.

Ethanolamine ammonia-lyase (EAL) is a 5’-deoxyadenosylcobalamin (AdoCbl; coenzyme B12) –dependent bacterial enzyme that catalyzes the deamination of the short-chain vicinal amino alcohols, aminoethanol and [S]- and [R]-2-aminopropanol. The coding sequence for EAL is located within the 17-gene eut operon, which codes for the broad spectrum of proteins that comprise the eut metabolosome sub-organelle structure. A high-resolution structure of the ~500 kDa EAL [(EutB-EutC)2]3 oligomer from Escherichia coli has been determined by X-ray crystallography, but high-resolution spectroscopic determinations of reactant intermediate state structures, and detailed kinetic and thermodynamic studies of EAL, have been conducted for the Salmonella typhimurium enzyme. Therefore, a statistically robust homology model for the S. typhimurium EAL is constructed from the E. coli structure. The model structure is used to describe the hierarchy of EutB and EutC subunit interactions that construct the native EAL oligomer, and specifically, to address the long-standing challenge of reconstitution of the functional oligomer from isolated, purified subunits. Model prediction that the (EutB2)3 oligomer assembly will occur from isolated EutB, and that this hexameric structure will template the formation of the complete, native [(EutB-EutC)2]3 oligomer, is verified by biochemical methods. Prediction that cysteine residues on the exposed subunit-subunit contact surfaces of isolated EutB and EutC will interfere with assembly by cystine formation is verified by activating effects of disulfide reducing agents. Ångstrom-scale congruence of the reconstituted and native EAL in the active site region is shown by electron paramagnetic resonance spectroscopy. Overall, the hierarchy of subunit interactions and microscopic features of the contact surfaces, that are revealed by the homology model, guide and provide a rationale for a refined genetic and biochemical approach to reconstitution of the functional [(EutB-EutC)2]3 EAL oligomer. The results establish a platform for further advances toward understanding the molecular mechanism of EAL catalysis, and for insights into therapy-targeted manipulation of the bacterial ethanolamine utilization (eut) metabolosome.

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn2+ to modulate the assembly kinetics and morphology of congeners of the amyloid β peptide (Aβ) associated with Alzheimer's disease. We now reveal a correlation among Aβ-Cu2+ coordination, peptide self-assembly, and neuronal viability. By using the central segment of Aβ, HHQKLVFFA or Aβ(13–21), which contains residues H13 and H14 implicated in Aβ-metal ion binding, we show that Cu2+ forms complexes with Aβ(13–21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Aβ(13–21)H14A, alters metal coordination, allowing Cu2+ to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Aβ can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Aβ self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

The transient decay reaction kinetics of the 1,1,2,2-2H4-aminoethanol generated CoII-substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been measured by using time-resolved, full-spectrum X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy in frozen aqueous solution over the temperature range of 190-207 K. The decay reaction involves sequential passage through the rearrangement step [substrate radical → product radical], and the step [product radical → diamagnetic product] that involves hydrogen atom transfer (HT) from carbon C5′ of the adenosine moiety of the cofactor to the product radical C2 center. As found for the 1H-substrate radical, the decay kinetics for the 2H-substrate radical over 190-207 K represent two non-interacting populations (fast decay population: normalized amplitude=0.44 ±0.07; observed rate constant, kobs,f=5.3×10−5 – 1.1×10−3 s−1; slow decay population: kobs,s=6.1×10−6 – 2.9×10−4 s−1). The 1H/2H isotope effects (IE) for the fast and slow decay reactions are 1.4 ±0.2 and 0.79 ±0.11, respectively. The IE on the fast phase is uniform over the temperature interval, and the value is consistent with an α-secondary hydrogen kinetic IE, which arises from changes in the force constants of the C-H bonds in the substrate radical structure, upon passing from the substrate radical state to the rearrangement transition state. Therefore, we propose that kobs,f represents the rate constant for the radical rearrangement, and that this step is the rate determining step in substrate radical decay. The Arrhenius activation energy for the 1H-substrate radical rearrangement (13.5 ±0.4 kcal/mol) is consistent with values from quantum chemical calculations performed on simple models. The results show that the core, radical rearrangement reaction is culled from the catalytic cycle in the low temperature system, thus establishing the system for detailed transient kinetic and spectroscopic analysis of protein structural and dynamic contributions to EAL catalysis.

Protein contributions to the substrate-triggered cleavage of the cobalt-carbon (Co-C) bond and formation of the cob(II)alamin-5′-deoxyadenosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved probing of the photoproduct dynamics by using ultraviolet-visible absorption spectroscopy on the 10−7 − 10−1 s time scale. Experiments were performed in a fluid dimethylsulfoxide/water cryosolvent system at 240 K, under conditions of kinetic competence for thermal cleavage of the Co-C bond in the ternary complex. The static ultraviolet-visible absorption spectra of holo-EAL and ternary complex are comparable, indicating that the binding of substrate does not labilize the cofactor cobalt-carbon (Co-C) bond by significantly distorting the equilibrium AdoCbl structure. Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5′-deoxyadenosyl radical pair quantum yields of <0.01 at 10−6 s in both holo-EAL and ternary complex. Three photoproduct states are populated following a saturating laser pulse, and labeled, Pf, Ps, and Pc. The relative amplitudes and first-order recombination rate constants of Pf (0.4-0.6; 40-50 s−1), Ps, (0.3-0.4; 4 s−1) and Pc (0.1-0.2; 0) are comparable in holo-EAL and in the ternary complex. Time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy shows that visible irradiation alters neither the kinetics of thermal cob(II)alamin-substrate radical pair formation, nor the equilibrium between ternary complex and cob(II)alamin-substrate radical pair, at 246 K. The results indicate that substrate binding to holo-EAL does not “switch” the protein to a new structural state, which promptly stabilizes the cob(II)alamin-5′-deoxyadenosyl radical pair photoproduct, either through an increased barrier to recombination, a decreased barrier to further radical pair separation, or lowering of the radical pair state free energy, or a combination of these effects. Therefore, we conclude that such a change in protein structure, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length, is not a basis of Co-C bond cleavage catalysis. The results suggest that, following the substrate trigger, the protein interacts with the cofactor to contiguously guide the cleavage of the Co-C bond, at every step along the cleavage coordinate, starting from the equilibrium configuration of the ternary complex. The cleavage is thus represented by a diagonal trajectory across a free energy surface, that is defined by chemical (Co-C separation) and protein configuration coordinates.

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5′-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.