bi0c00027_si_001.pdf (3.05 MB)
Biophysical Techniques for Distinguishing Ligand Binding Modes in Cytochrome P450 Monooxygenases
journal contribution
posted on 2020-02-21, 21:45 authored by Matthew
N. Podgorski, Joshua S. Harbort, Tom Coleman, Jeanette E. Stok, Jake A. Yorke, Luet-Lok Wong, John B. Bruning, Paul V. Bernhardt, James J. De Voss, Jeffrey R. Harmer, Stephen G. BellThe
cytochrome P450 superfamily of heme monooxygenases catalyzes
important chemical reactions across nature. The changes in the optical
spectra of these enzymes, induced by the addition of substrates or
inhibitors, are critical for assessing how these molecules bind to
the P450, enhancing or inhibiting the catalytic cycle. Here we use
the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted
benzoic acids to investigate different binding modes. 4-Methoxybenzoic
acid elicits an archetypal type I spectral response due to a ≥95%
switch from the low- to high-spin state with concomitant dissociation
of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic
acid induced different type II ultraviolet–visible (UV–vis)
spectral responses in CYP199A4. The former induced a greater red shift
in the Soret wavelength (424 nm vs 422 nm) along with a larger overall
absorbance change and other differences in the α-, β-,
and δ-bands. There were also variations in the ferrous UV–vis
spectra of these two substrate-bound forms with a spectrum indicative
of Fe–N bond formation with 4-(pyridin-3-yl)benzoic acid. The
crystal structures of CYP199A4, with the pyridinyl compounds bound,
revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is
coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua
ligand remains. Continuous wave and pulse electron paramagnetic resonance
data in frozen solution revealed that the substrates are bound in
the active site in a form consistent with the crystal structures.
The redox potential of each CYP199A4–substrate combination
was measured, allowing correlation among binding modes, spectroscopic
properties, and the observed biochemical activity.