Abstract
Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. Here we demonstrate the application of this method using fungal NO reductase, a heme-containing enzyme, at room temperature. Twenty milliseconds after caged-NO photolysis, we identify a NO-bound form of the enzyme, which is an initial intermediate with a slightly bent Fe-N-O coordination geometry at a resolution of 2.1 Å. The NO geometry is compatible with those analyzed by XFEL-based cryo-crystallography and QM/MM calculations, indicating that we obtain an intact Fe3+-NO coordination structure that is free of X-ray radiation damage. The slightly bent NO geometry is appropriate to prevent immediate NO dissociation and thus accept H− from NADH. The combination of using XFEL and a caged-compound is a powerful tool for determining functional enzyme structures during catalytic reactions at the atomic level.
Highlights
Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions
We demonstrate a TR-SFX experiment in conjunction with a caged-compound as a reaction trigger, using nitric oxide reductase isolated from the fungus Fusarium oxysporum (P450nor)
In the present study, we demonstrated the successful application of a caged-compound for TR-SFX to characterize the ferric nitric oxide (NO) complex structure, an initial intermediate of the P450nor reaction at ambient temperature
Summary
Time-resolved serial femtosecond crystallography using an X-ray free electron laser (XFEL) in conjunction with a photosensitive caged-compound offers a crystallographic method to track enzymatic reactions. A new experimental technique using XFELs is serial fs crystallography (SFX), in which single-shot diffraction images are collected in series from a continuous flow of micro-crystals with random orientation[1, 2]. The combination of caged-substrates with the pump–probe TR-SFX technique is another crystallographic method that may be used to examine enzymatic reactions at ambient temperature. We demonstrate a TR-SFX experiment in conjunction with a caged-compound as a reaction trigger, using nitric oxide reductase (nor) isolated from the fungus Fusarium oxysporum (P450nor). Because N2O is the main ozone-depleting substance and a greenhouse gas[20], the NO reduction reaction mechanism that produces troscopic
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