NMR Studies of Ligand Binding to P450_eryF Provides Insight into the Mechanism of Cooperativity

Journal Article · Wed Feb 01 00:00:00 EST 2006 · Biochemistry

DOI:https://doi.org/10.1021/bi0518895· OSTI ID:1152355

Roberts, Arthur G. ^[1]; Diaz, M. Delores ^[1]; Lampe, Jed N. ^[1]; Shireman, Laura M. ^[1]; Grinstead, Jeffrey S. ^[1]; Dabrowski, Michael J. ^[1]; Pearson, Josh T. ^[1]; Bowman, Michael K. ^[2]; Atkins, William M. ^[1]; Campbell, A. Patricia ^[1]

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1. Univ. of Washington, Seattle, WA (United States)
2. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

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 heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still 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 P450_eryF. UV/vis absorbance spectra of P450_eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of KS = 4 and 200 μM, respectively. The broadening and shifting observed in the 2D-{¹H,¹⁵N}-HSQC-monitored titrations of ¹⁵N-Phe-labeled P450_eryF with 9-AP and TST indicated binding on 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 same in each titration, suggesting that the two ligands ''contact'' the same phenylalanines within the active site of P450_eryF. This finding is in agreement with X-ray crystal structures of bound P450_eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450_eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.

• Research Organization: Environmental Molecular Sciences Laboratory (EMSL), Richland, WA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• OSTI ID: 1152355
• Journal Information: Biochemistry, Vol. 45, Issue 6; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

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