Is There a Dynamic Protein Contribution to the Substrate Trigger in Coenzyme B₁₂‐Dependent Ethanolamine Ammonia Lyase?

Abstract

“Cohort‐ry” in motion: The chemistry following CoC bond homolysis in coenzyme B12‐dependent ethanolamine ammonia lyase is known to favor dissociation, but what of the protein contribution? Experiments reveal the radical pair reaction dynamics to be coupled to the ps–ns protein dynamics in B12 photolysis. This raises the possibility of a subtle, dynamic contribution to homolysis, which acts in cohort with electrostatics and H‐abstraction from the substrate.

Protein Motions Are Coupled to the Reaction Chemistry in Coenzyme B12‐Dependent Ethanolamine Ammonia Lyase

“…Consequently, the principle kinetics from single value decomposition analysis only fit to the sum of two exponentials as opposed to the four exponential fit to the AdoCbl data (see Figure S1 and Table S1 in the Supporting Information). Although protein binding is known to affect the kinetics of AdoCbl photolysis, [7,30,31] the most marked effect is on the excited state processes, which have not been fully resolved here. However, values for k 3 and k 4 -which represent radical pair formation and dynamics-are of the same order across the available TRIR and UV/Vis data sets (see Table S1 in the Supporting Information).…”

“…Such an interaction is consistent with the viscosity dependence of the recombination kinetics after photolysis in EAL, recently attributed to a mobile active-site residue (likely Glu 287 ) [18] interacting with the adenosyl radical. [7] Whether the TRIR peaks at 1650 and 1661 cm À1 are from the protein or otherwise, they are a relatively minor component of the signal, the majority of which resembles the predicted changes in the cofactor that accompany homolysis (calculated using DFT). [20] The fact that such cofactor signals are not clearly evident above the noise in the SF-FTIR spectra suggest that these data are dominated by signals representing changes in the protein following substrate binding or turnover.…”

“…[18] Instead, Robertson et al have proposed a more subtle, cooperative effect from the protein, [19] and, in support of this, we have recently published evidence that implicates a dynamic contribution from an active-site residue. [7] EAL exhibits three broad amide absorptions in the mid-IR spectrum (Figure 1 a, solid line) with the amide I band (1625-1675 cm À1 ) representing the most intense signal. AdoCbl also contributes to the signal in this region (Figure 1 a, dashed line), with a broad band centered around Scheme 1.…”

“…[1,2] Remarkably, thermal homolysis rates achieved by these enzymes are about 10 12 times greater than that of the free cofactor in solution. [3][4][5][6] Recent data indirectly suggest protein motions may be coupled to the reaction chemistry, [7] a hotly debated issue in modern enzymology. [8,9] Herein, from studies using both laser flash photolysis and stoppedflow infrared (IR) techniques, we present the first direct evidence for a protein motion that correlates with coupled CoÀC bond homolysis and H abstraction from the substrate in ethanolamine ammonia lyase (EAL) from Salmonella enterica.…”

“…[7] After CoÀC homolysis, the adenosyl radical abstracts hydrogen from ethanolamine, followed by radical rearrangement and dissociation to products (Scheme 1). [10,11] In theory, the only steps resolvable by UV/Vis spectroscopy are the interconversion of cob(III)alamin and cob(II)alamin at the beginning and end of turnover.…”