Capturing an initial intermediate during the P450nor enzymatic reaction using time-resolved XFEL crystallography and caged-substrate

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.

Fig. 1

Reaction cycle of P450nor. P450nor reduces NO to N₂O through the NO-bound state and intermediate-I

Fig. 2

Caged-NO photolysis. One caged-NO releases two NO molecules upon UV light illumination

Fig. 3

Crystal spectroscopy of ferric P450nor. TR-visible absorption difference spectra of a MC-1 and b MC-2 after caged-NO photolysis. The difference was calculated by subtracting the spectrum recorded prior to photolysis. c Static IR spectrum of MC-2 after caged-NO photolysis. All the measurements were performed in the presence of the SFX carrying medium (hydroxyethyl cellulose matrix) at 293 K

Fig. 4

SFX structures of P450nor. a Resting-state structure. b, c Transient structures at 20 ms after caged-NO photolysis in the b absence and c presence of NADH. The 2F _o−F _c maps are shown in gray and contoured at 1.2σ. The F _o−F _c map is shown in orange and contoured at 4.0σ in a, whereas the F _o(“Light”)−F _o(“Dark2”) difference Fourier maps are shown in turquoise (positive) and magenta (negative) and contoured at 6.5σ in b and 3.2σ in c. All data were taken at ambient temperature. In a, the structure using the “Dark2” data of MC-2 is presented

Fig. 5

Structure of the NADH channel entrance in the a A and b B chains of resting P450nor at ambient temperature. The 2F _o−F _c maps are shown in gray and contoured at 1.0σ. The F _o−F _c positive maps are shown in turquoise and contoured at 2.3σ. The structure factor F _c was calculated from the open form (blue Cα trace) of the NADH channel. The refined structure of the closed form is shown in orange (40% occupancy). The structures using the “Dark2” data of MC-2 are presented

Fig. 6

Structures of the ferric NO complex of P450nor obtained by a SF-ROX and b, c synchrotron X-ray crystallography. The X-ray doses in the synchrotron data collection were b 0.72 and c 5.7 MGy. The 2F _o−F _c maps are shown in gray and contoured at 1.2σ, and the F _o−F _c positive maps are shown in turquoise and contoured at 7.0σ. All data were taken at 100 K in the absence of NADH

Fig. 7

Potential energy surfaces for Fe-N-O bending. The surfaces of the QM/MM, reduced QM/MM, and isolated heme QM models are shown by blue circles, magenta squares, and cyan triangles, respectively