Structural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase

Abstract

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.

Fig. 1. Overall structure of CYP116B46.

The polypeptide structure is presented as a cartoon model, with the three domains and connecting linkers in different colors. The heme domain, reductase domain and ferredoxin domain are colored in green, magenta and cyan. The bound ligands that are shown in stick models are labeled, and so are the N- and C-terminus. The lower panel shows the primary structure of CYP116B46 with the amino acid numbers indicated for each region.

Fig. 2. Intramolecular electron transfer pathway.

a The CYP116B46 structure is presented as in Fig. 1. The NADPH modeled from pea FNR is displayed as white stick. The thick arrows indicate the direction of electron transfer and the dashed arrow represents the putative portal for substrate entry. Dashed lines connect the redox centers observed in the structure and the corresponding distances (in Å) are also indicated. Black and orange dashed lines represent edge-to-edge distance between heme and [2Fe-2S] cluster and [2Fe-2S] cluster and FMN, respectively. b A close-up view of (a). The electron transfer path between the C8 methyl group of FMN and the Fe2 of [2Fe-2S] cluster is indicated by a dashed line. The protein residues that may constitute the long-range electron transfer path between [2Fe-2S] and heme are shown as sticks. Residues that may contribute both main chain and side chain to the electron delivery path are noted by asterisks and their main chains are displayed as spheres. Residues colored in green and cyan belong to the heme and ferredoxin domain, respectively. The orange dashed line indicates the distance between FMN C8 methyl group and [2Fe-2S] cluster Fe2 atom.

Fig. 3. Uncovering the hidden route.

a The activity of Ala variants of several residues that are located between [2Fe-2S] and heme was measured. Triplicate or quadruple assay was performed in each independent experiment and the average (mean) and standard error (standard deviation, s.e.m.) for each group were calculated. Three independent experiments were conducted and similar results were yielded. The relative enzyme activity of each variant is presented as a percentage of the wild-type enzyme. The corresponding data points are displayed as dots. The sample size is 4 for WT, R392A, R718A, E723A, and E729A, and is 3 for F378A, R388A, Q725A, and S726A. UD, undetectable. Asterisk indicates that F378A lacked CO-binding capacity. Source data are provided as a Source Data file. The residues that may play a role in enzyme activity are highlighted and shown in (b). Residue F378 and C385 that may be required to bind to the heme are colored in black. The labels and color schemes of (b) are the same as in Fig. 2b.