| Abstract
| - Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins.Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropiccooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery arestill not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR)spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450eryF.UV/vis absorbance spectra of P450eryF demonstrated that binding occurs with apparent negative homotropiccooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-deriveddissociation constants of KS = 4 and 200 μM, respectively. The broadening and shifting observed in the2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450eryF with 9-AP and TST indicated bindingon intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hill-equation-derived KS values for these two ligands. Regardless of the type of spectral perturbation observed(broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the samein each titration, suggesting that the two ligands “contact” the same phenylalanines within the active siteof P450eryF. This finding is in agreement with X-ray crystal structures of bound P450eryF showing differentligands occupying similar active-site niches. Complex spectral behavior was additionally observed for asmall collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450eryF conformational substates. A structural and energeticmodel is presented that combines the energetics and structural aspects of 9-AP and TST binding derivedfrom these observations.
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