Abstract
Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We recently discovered an unusually slow reaction at room temperature that occurs on the order of days: the in crystalline reverse oxidative decay of the chemically labile (6S)-5,6,7,8-tetrahydrofolate in complex with its producing enzyme Escherichia coli dihydrofolate reductase. Here, we report the critical analysis of a representative dataset at an intermediate reaction time point. A quinonoid-like intermediate state lying between tetrahydrofolate and dihydrofolate features a near coplanar geometry of the bicyclic pterin moiety, and a tetrahedral sp3 C6 geometry is proposed based on the apparent mFo-DFc omit electron densities of the ligand. The presence of this intermediate is strongly supported by Bayesian difference refinement. Isomorphous Fo-Fo difference map and multi-state refinement analyses suggest the presence of end-state ligand populations as well, although the putative intermediate state is likely the most populated. A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin. We postulate that the constraints on the ligand imposed by the interactions with the protein environment might be the origin of the slow reaction observed by time-resolved crystallography.
Highlights
Time-resolved crystallography is an experimental technique that can detect molecular changes at atomic and temporal resolutions.[1]
A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin
When the structure is refined with a single ligand [using previously reported binary tetrahydrofolate complex (PDB) Ligand THG, (6S)-5,6,7,8-tetrahydrofolate], the pterin rings of the ligand appear to be near coplanar as compared to the puckered rings in the tetrahydrofolate bound complex
Summary
Time-resolved crystallography is an experimental technique that can detect molecular changes at atomic and temporal resolutions.[1] Due to the feasibility of rapid reaction initiation by a laser pulse, this technique has been widely used to study light-active protein systems including myoglobin,[2] hemoglobin,[3] photoactive yellow protein,[4,5,6] photosystem II,[7,8,9] and rhodopsin.[10] Recent advances in sample delivery systems and femtosecond X-ray free electron lasers (XFELs) have allowed time-resolved serial femtosecond crystallography (TR-SFX) to be extended to other systems as well.[1,11,12,13] For example, the mixand-inject method can rapidly and uniformly initiate an enzymatic reaction[11,12] or an RNA-ligand interaction[13] in micro/nanocrystals before diffraction whose rate is limited by diffusion. The implications of the current observation on the molecular mechanism of tetrahydrofolate to dihydrofolate conversion and its possible generalization to other systems are discussed
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