Search

Cytochromes P450 (CYPs) are heme-containing enzymes that are present in all kingdoms of life and share a structurally homologous, globular protein fold. CYPs utilize structures distal to the heme to recognize and coordinate substrates, while the necessary interactions with redox partner proteins are mediated at the opposite, proximal surface. In the current study, we investigated the functional allostery across the heme for the bacterial enzyme CYP121A1, which utilizes a non-polar distal-to-distal dimer interface for specific binding of its dicyclotyrosine substrate. Fluorine-detected Nuclear Magnetic Resonance (19F-NMR) spectroscopy was combined with site-specific labeling of a distal surface residue (S171C of the FG-loop), one residue of the B-helix (N84C), and two proximal surface residues (T103C and T333C) with a thiol-reactive fluorine label. Adrenodoxin was used as a substitute redox protein and was found to promote a closed arrangement of the FG-loop, similar to the addition of substrate alone. Disruption of the protein–protein interface by mutagenesis of two CYP121 basic surface residues removed the allosteric effect. Moreover, 19F-NMR spectra of the proximal surface indicate that ligand induced allostery modulates the environment at the C-helix but not the meander region of the enzyme. In light of the high degree of structural homology in this family of enzymes, we interpret the findings from this work to represent a conserved allosteric network in CYPs. [ABSTRACT FROM AUTHOR]

Cytochromes P450 are versatile enzymes that function in endobiotic and xenobiotic metabolism and undergo meaningful structural changes that relate to their function. However, the way in which conformational changes inform the specific recognition of the substrate is often unknown. Here, we demonstrate the utility of fluorine (19F)-NMR spectroscopy to monitor structural changes in CYP121A1, an essential enzyme from Mycobacterium tuberculosis. CYP121A1 forms functional dimers that catalyze the phenol-coupling reaction of the dipeptide dicyclotyrosine. The thiol-reactive compound 3-bromo-1,1,1-trifluoroacetone was used to label an S171C mutation of the enzyme FG loop, which is located adjacent to the homodimer interface. Substrate titrations and inhibitor-bound 19F-NMR spectra indicate that ligand binding reduces conformational heterogeneity at the FG loop in both the dimer and in an engineered monomer of CYP121A1. However, only the dimer was found to promote a substrate-bound conformation that was preexisting in the substrate-free spectra, thus confirming a role for the dimer interface in dicyclotyrosine recognition. Moreover, 19F-NMR spectra in the presence of substrate analogs indicate the hydrogen-bonding feature of the dipeptide aromatic side chain as a dicyclotyrosine specificity criterion. This study demonstrates the utility of 19F-NMR as applied to a multimeric cytochrome P450, while also revealing mechanistic insights for an essential M. tuberculosis enzyme. [ABSTRACT FROM AUTHOR]

Human Ferredoxin 1, also referred to as Adrenodoxin (Adx), is the sole electron carrier supporting the function of all seven mitochondrial cytochrome P450 (CYP) enzymes. Adx utilizes conserved negatively charged residues along its α-helix3 to interact with either the proximal surface of CYP enzymes or the binding surface of Adrendodoxin Reductase (AdR). However, in the oxidized state, Adx assumes a monomer-homodimer equilibrium that requires the presence of its unstructured C-terminal tail. Crystallographic structures of full-length human Adx dimers indicate that part of the binding surface necessary for its interactions with CYPs or with AdR is partially occluded by the dimer interface. In this study, protein NMR spectroscopy was used to interrogate the interactions between full-length (2–124) or truncated monomeric (2–108) human Adx and human CYP24A1 (with and without its vitamin-D substrate) as well as interactions with AdR. Here, monomeric Adx induced a similar pattern of peak broadening as that induced by addition of CYP24A1 substrate, consistent with a 1:1 Adx:CYP interaction as the functional complex. Additionally, removal of the C-terminal tail appears to enhance the interaction with AdR, despite removal of some of the AdR contacts in the tail region. This finding was also supported by an NMR competition assay. These findings suggest that the Adx dimers do not undergo meaningful interactions with either CYP or AdR, but may instead be responsible for regulating access to monomeric Adx. These conclusions are discussed in the context of a revised model of the Adx electron shuttle mechanism. Adx-detected NMR data in this study support the formation of mutually exclusive complexes between Adx-Adx, Adx-CYP, or Adx-AdR during electron delivery. The conserved ⍺ helix 3 (highlighted in red) is necessary for CYP and AdR interactions, but is unavailable in Adx homodimers. [Display omitted] • Truncation of the Adx C-terminal tail regulates its protein interactions. • CYP24A1 vitamin-D substrate preferences the 1:1 complex with human Adx. • Truncated (monomeric) Adx appears to bind AdR stronger than WT Adx. • NMR data support occlusion of the Adx ⍺ helix 3 by the Adx:Adx dimer interface. • Adx forms mutually exclusive complexes with Adx, CYP, and AdR. [ABSTRACT FROM AUTHOR]