7 research outputs found
Conformational Change Induced by Putidaredoxin Binding to Ferrous CO-ligated Cytochrome P450cam Characterized by 2D IR Spectroscopy
The importance of conformational dynamics to protein function is now well-appreciated. An outstanding question is whether they are involved in the effector role played by putidaredoxin (Pdx) in its reduction of the O2 complex of cytochrome P450cam (P450cam), an archetypical member of the cytochrome P450 superfamily. Recent studies have reported that binding of Pdx induces a conformational change from a closed to an open state of ferric P450cam, but a similar conformational change does not appear to occur for the ferrous, CO-ligated enzyme. To better understand the effector role of Pdx when binding the ferrous, CO-ligated P450cam, we applied 2D IR spectroscopy to compare the conformations and dynamics of the wild-type (wt) enzyme in the absence and presence of Pdx, as well as of L358P P450cam (L358P), which has served as a putative model for the Pdx complex. The CO vibrations of the Pdx complex and L358P report population of two conformational states in which the CO experiences distinct environments. The dynamics among the CO frequencies indicate that the energy landscape of substates within one conformation are reflective of the closed state of P450cam, and for the other conformation, differ from the free wt enzyme, but are equivalent between the Pdx complex and L358P. The two states co-populated by the Pdx complex are postulated to reflect a loosely bound encounter complex and a more tightly bound state, as is commonly observed for the dynamic complexes of redox partners. Significantly, this study shows that the binding of Pdx to ferrous, CO-ligated P450cam does perturb the conformational ensemble in a way that might underlie the effector role of Pdx
Dynamics and Conformational Heterogeneity in Cytochrome P450s via Infrared Spectroscopy
Cytochrome P450s (P450s) are a superfamily of enzymes that catalyze oxidation of unactivated hydrocarbons. However, the means by which P450s control (1) regioselectivity of their activity and (2) specificity in their molecular recognition remain largely elusive. Toward investigation of the role of dynamics in the regioselectivity of the archetypal cytochrome P450cam (P450cam), two-dimensional infrared spectroscopy has been applied with heme-bound carbon monoxide (CO) as an infrared probe of the active site. The data support a model for P450cam regioselectivity in which binding of different substrates to P450cam variably stabilizes the active site into two distinct states, each associated with different dynamics linked to different levels of regioselectivity. To investigate the role of conformational heterogeneity in P450cam substrate specificity, infrared spectoscopy was combined with the site-specific incorporation of nitrile probes at distinct P450cam microenvironments. This approach enabled differentiation of changes experienced at each of those environments when d-camphor and/or CO binds to the active site. Finally, the impact of conformational heterogeneity on the affinity of substrate molecular recognition by wild-type and mutant P450cam was evaluated using both CO and nitrile probes. This study suggests that the nature of the conformations populated in the unbound states influences the affinity for different substrates. Collectively, these studies provide new insight into the roles of conformational heterogeneity and dynamics in P450cam activity. Furthermore, these studies help to lay the foundation for efforts toward understanding the roles of conformational heterogeneity and dynamics in the function of human P450s, for which unraveling the mechanisms involved in Phase I metabolism is a topic of great pharmacological concern
Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy
Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function. © 2016 American Chemical Society1991sciescopu
Conformational Landscape and the Selectivity of Cytochrome P450cam
Conformational
heterogeneity and dynamics likely contribute to
the remarkable activity of enzymes but are challenging to characterize
experimentally. These features are of particular interest within the
cytochrome P450 class of monooxygenases, which are of great academic,
medicinal, and biotechnological interest as they recognize a broad
range of substrates, such as various lipids, steroid precursors, and
xenobiotics, including therapeutics. Here, we use linear and 2D IR
spectroscopy to characterize the prototypical P450, cytochrome P450cam,
bound to three different substrates, camphor, norcamphor, or thiocamphor,
which are hydroxylated with high, low, and intermediate regioselectivity,
respectively. The data suggest that specific interactions with the
substrate drive the population of two different conformations, one
that is associated with high regioselectivity and another associated
with lower regioselectivity. Although Y96 mediates a hydrogen bond
thought necessary to orient the substrate for high regioselectivity,
the population and dynamics of the conformational states are largely
unaltered by the Y96F mutation. This study suggests that knowledge
of the conformational landscape is central to understanding P450 activity,
which has important practical ramifications for the design of therapeutics
with optimized pharmacokinetics, and the manipulation of P450s, and
possibly other enzymes, for biotechnological applications
Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy
Conformational changes are central
to protein function but challenging
to characterize with both high spatial and temporal precision. The
inherently fast time scale and small chromophores of infrared (IR)
spectroscopy are well-suited for characterization of potentially rapidly
fluctuating environments, and when frequency-resolved probes are incorporated
to overcome spectral congestion, enable characterization of specific
sites in proteins. We selectively incorporated <i>p</i>-cyanophenylalanine
(CNF) as a vibrational probe at five distinct locations in the enzyme
cytochrome P450cam and used IR spectroscopy to characterize the environments
in substrate and/or ligand complexes reflecting those in the catalytic
cycle. Molecular dynamics (MD) simulations were performed to provide
a structural basis for spectral interpretation. Together the experimental
and simulation data suggest that the CN frequencies are sensitive
to both long-range influences, resulting from the particular location
of a residue within the enzyme, as well as short-range influences
from hydrogen bonding and packing interactions. The IR spectra demonstrate
that the environments and effects of substrate and/or ligand binding
are different at each position probed and also provide evidence that
a single site can experience multiple environments. This study illustrates
how IR spectroscopy, when combined with the spectral decongestion
and spatial selectivity afforded by CNF incorporation, provides detailed
information about protein structural changes that underlie function