Heads and tails: Energetic and structural bases of binding and electron transfer performance of cofactors at the Q(A) site of the reaction center protein from Rhodobacter sphaeroides

Factors controlling cofactor binding and electron transfer function at the primary quinone, Q\sb{\rm A} site in isolated reaction center protein from Rhodobacter sphaeroides are determined from effects of quinone cofactor head group and tail structure alterations on: (1) Q\sb{\rm A} site binding free energy in water (\rm\Delta G\sbsp{\rm B,W}{\circ}) and hexane (\rm\Delta G\sbsp{\rm B,H}{\circ}), (2) cofactor electrochemical potential in the site relative to dimethylformamide solution, and (3) temperature and reaction free energy dependences of the electron transfer rate constants for Q\sb{\rm A} reduction by bacteriopheophytin (k\sb1) and charge recombination with the oxidized bacteriochlorophyll dimer (k\sb{\rm b}). A thermodynamic cycle formalism is developed to resolve intrinsic ligand-protein interaction and aqueous solvation contributions to binding. Relative to hexane, the aqueous solvation contribution to \rm\Delta G\sbsp{\rm B,W}{\circ} is specified by 0.8\rm\Delta G\sbsp{\rm tr}{\circ}, where \rm\Delta G\sbsp{\rm tr}{\circ} is the quinone solvent transfer free energy (range: -0.5 to -6.8 Kcal/mole). Effects of systematic variation of the native isoprene tail structure on \rm\Delta G\sbsp{\rm B,H}{\circ} and \rm\Delta G\sbsp{\rm B,W}{\circ} at the Q\sb{\rm A} and secondary, Q\sb{\rm B} sites reveals: (1) a defined tail binding domain spanning the first three isoprene units, and (2) a strong binding specificity for the isoprene relative to saturated alkyl structures ($\u3e$4.1 Kcal/mole). Tail structure does not significantly influence electron transfer rates (variation of k\sb1 and k\sb{\rm b} $\u3c$5 fold). Comparison of Q\sb{\rm A} site \rm\Delta G\sbsp{\rm B,H}{\circ} values of quinone and analog head groups in which one or both carbonyl groups are removed shows that one carbonyl oxygen atom dominates hydrogen bond contact with the protein ($\rm\Delta H$ = -3.5 Kcal/mole). In contrast, both oxygen atoms participate in the semiquinone-site interaction, as shown by a 166 mV loss of in situ cofactor redox couple stability relative to DMF associated with single removal. The rationally-selected exotic cofactors tetrafluoro- and trinitro-fluorenone, and m-dinitrobenzene display k\sb1 and k\sb{\rm b} dependences on temperature (7 to 295 K) and reaction free energy (\rm\Delta G\sbsp{\rm et}{\circ}) that are comparable with quinones. These results indicate that: (1) structural elements of the native quinone-Q\sb{\rm A} site interaction are not essential for electron transfer function, and (2) the values of k\sb1 and k\sb{\rm b} appear to be determined at the Q\sb{\rm A} site primarily through the contribution of the in situ electrochemical free energy of the cofactor to \rm\Delta G\sbsp{\rm et}{\circ}

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

Adenosylcobalamin-dependent enzymes accelerate the cleavage of the cobalt–carbon (Co–C) bond of the bound coenzyme by >10¹⁰-fold. The cleavage-generated 5′-deoxyadenosyl radical initiates the catalytic cycle by abstracting a hydrogen atom from substrate. Kinetic coupling of the Co–C bond cleavage and hydrogen-atom-transfer steps at ambient temperatures has interfered with past experimental attempts to directly address the factors that govern Co–C bond cleavage catalysis. Here, we use time-resolved, full-spectrum electron paramagnetic resonance spectroscopy, with temperature-step reaction initiation, starting from the enzyme–coenzyme–substrate ternary complex and ²H-labeled substrate, to study radical pair generation in ethanolamine ammonia-lyase from <i>Salmonella typhimurium</i> at 234–248 K in a dimethylsulfoxide/water cryosolvent system. The monoexponential kinetics of formation of the ²H- and ¹H-substituted substrate radicals are the same, indicating that Co–C bond cleavage rate-limits radical pair formation. Analysis of the kinetics by using a linear, three-state model allows extraction of the microscopic rate constant for Co–C bond cleavage. Eyring analysis reveals that the activation enthalpy for Co–C bond cleavage is 32 ± 1 kcal/mol, which is the same as for the cleavage reaction in solution. The origin of Co–C bond cleavage catalysis in the enzyme is, therefore, the large, favorable activation entropy of 61 ± 6 cal/(mol·K) (relative to 7 ± 1 cal/(mol·K) in solution). This represents a paradigm shift from traditional, enthalpy-based mechanisms that have been proposed for Co–C bond-breaking in B₁₂ enzymes. The catalysis is proposed to arise from an increase in protein configurational entropy along the reaction coordinate

Mesodomain and Protein-Associated Solvent Phases with Temperature-Tunable (200–265 K) Dynamics Surround Ethanolamine Ammonia-Lyase in Globally Polycrystalline Aqueous Solution Containing Dimethyl Sulfoxide

Electron paramagnetic resonance spectroscopy of the spin probe, TEMPOL, is used to resolve solvent phases that surround the ethanolamine ammonia-lyase (EAL) protein from Salmonella typhimurium at low temperature (<i>T</i>) in frozen, globally polycrystalline aqueous solution and to report on the <i>T</i> dependence of their detectably rigid and fluid states. EAL plays a role in human gut microbiome-based disease conditions, and physicochemical studies provide insight into protein structure and mechanism, toward potential therapeutics. Temperature dependences of the rotational correlation times (τ_c; detection range, 10^–11 ≤ τ_c ≤ 10^–7 s) and the corresponding weights of TEMPOL tumbling components from 200 to 265 K in the presence of EAL are measured in two frozen systems: (1) water-only and (2) 1% v/v dimethyl sulfoxide (DMSO). In the water-only condition, a protein-vicinal solvent component detectably fluidizes at 230 K and melts the surrounding ice-crystalline region with increasing <i>T</i>, creating a bounded, relatively high-viscosity aqueous solvent domain, up to 265 K. In the EAL, 1% v/v DMSO condition, two distinct concentric solvent phases are resolved around EAL: protein-associated domain (PAD) and mesodomain. The DMSO aqueous mesodomain fluidizes at 200 K, followed by PAD fluidization at 210 K. The interphase dynamical coupling is consistent with the spatial arrangement and significant contact areas of the phases, indicated by the experimentally determined mean volume ratio, <i>V</i>(mesodomain)/<i>V</i>(PAD)/<i>V</i>(protein) = 0.5:0.3:1.0. The results provide a rationale for native chemical reactions of EAL at <i>T</i> < 250 K and an advance toward precise control of solvent dynamics as a tunable parameter for quantifying the coupling between solvent and protein fluctuations and chemical reaction steps in EAL and other enzymes