32 research outputs found
Structure and Thermal Behaviour of Dichlorobis(thiourea)cadmium(II), a Single-Source Precursor for CdS Thin Films.
Synthetic, Thermodynamic and Crystallographic Studies of Pentacoordinated Copper(II) Complexes with [24.31]Adamanzane, 1,4,7,10-Tetraazabicyclo[5.5.3]pentadecane, and Bromide, Iodide, Hydroxide, Water or Ammonia.
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 (4.1 Kcal/mole). Tail structure does not significantly influence electron transfer rates (variation of k\sb1 and k\sb{\rm b} 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 ( = -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}
Interaction of the Substrate Radical and the 5‘-Deoxyadenosine-5‘-Methyl Group in Vitamin B 12
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 >10<sup>10</sup>-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 <sup>2</sup>H-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 <sup>2</sup>H- and <sup>1</sup>H-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 B<sub>12</sub> 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<sub>c</sub>; detection range, 10<sup>–11</sup> ≤ τ<sub>c</sub> ≤ 10<sup>–7</sup> 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