19 F NMR Illustrates Novel Aspects of Cytochrome P450 Systems

Abstract

Understanding conformational dynamics is an integral part of understanding function in cytochrome P450 enzymes (CYPs), a ubiquitous class of heme-coordinated enzymes that are important for xenobiotic and endogenous metabolism. While nuclear magnetic resonance (NMR) is well suited to detect changes in the structures of CYPs and their associated redox partners, use of traditional isotopic labels are difficult to apply to such large enzymes and complexes. In this work, we have employed a thiol-reactive 19F label, 3-bromo-1,1,1-trifluoracetone (BTFA), to probe various aspects of Class-I CYP systems in a targeted approach. 19F is well suited for these studies due in part to its enhanced sensitivity over traditional NMR observable nuclei, as well as the fact that it does not naturally occur in proteins, which reduces the overall complexity of the resulting spectra. Here we deploy this strategy to study different aspects of CYP function, including details of redox partner interaction in a mammalian CYP and dynamics of substrate binding in a bacterial CYP. Our lab has previously used traditional 15N NMR to study the interaction between the vitamin D metabolizing enzyme, CYP24A1, and its redox partner, adrenodoxin (Adx). Using labeled Adx, we were able to titrate unlabeled CYP24A1 and monitor changes in the spectra, but were limited by the extent of signal loss attributed to the size of the complex. Substituting 15N labeling with two selectively placed 19F probes on α-helix 1 and α-helix 3 of Adx, we were able to probe biologically relevant complexes of these enzymes. Upon addition of CYP24A1 to 19F labeled Adx, we observe differential line broadening on α-helix 3, consistent with other studies implicating this region in CYP recognition. We also observe changes in the heterogeneity of the signal at α-helix 3, suggesting that this site undergoes conformational selection upon binding. The same probe was also applied to study the solution structure of CYP121 from Mycobacterium tuberculosis. Crystal structures of CYP121 show ligands clustered to a pocket near the F-G helices, which may play an important role in substrate recognition and control access to the active site. A probe was attached to the loop connecting these two helices (FG-loop) and monitored during titration with the substrate, cyclodityrosine (cYY). The 19F NMR spectrum for the FG-loop is heterogeneous suggesting multiple conformations exist in solution, but converges to a single peak upon cYY addition, representing a bound form. Utilizing an engineered monomer, our data also suggest that the dimer interface confers structural rigidity at this site, and in the dimeric form a population of enzyme exists in the ‘bound’ conformation prior to ligand introduction. Interestingly, the ratios of CYP121:cYY that are required to saturate the observed NMR response do not match with ratios required to detect binding using other techniques, thereby suggesting the 19F probe on the FG-loop is monitoring a ‘pre-catalytic’ binding event. These findings highlight the utility of employing an underutilized NMR label to understand function in an important class of enzymes.