3site Multisubstrate-Bound State of Cytochrome P450cam

Abstract

We identified a multisubstrate-bound state, hereby referred as a 3site state, in cytochrome P450cam via integrating molecular dynamics simulation with nuclear magnetic resonance (NMR) pseudocontact shift measurements. The 3site state is a result of simultaneous binding of three camphor molecules in three locations around P450cam: (a) in a well-established "catalytic" site near heme, (b) in a kink-separated "waiting" site along channel-1, and (c) in a previously reported "allosteric" site at E, F, G, and H helical junctions. These three spatially distinct binding modes in the 3site state mutually communicate with each other via homotropic allostery and act cooperatively to render P450cam functional. The 3site state shows a significantly superior fit with NMR pseudo contact shift (PCS) data with a Q-score of 0.045 than previously known bound states and consists of D251 free of salt-bridges with K178 and R186, rendering the enzyme functionally primed. To date, none of the reported cocomplex of P450cam with its redox partner putidaredoxin (pdx) has been able to match solution NMR data and controversial pdx-induced opening of P450cam's channel-1 remains a matter of recurrent discourse. In this regard, inclusion of pdx to the 3site state is able to perfectly fit the NMR PCS measurement with a Q-score of 0.08 and disfavors the pdx-induced opening of channel-1, reconciling previously unexplained remarkably fast hydroxylation kinetics with a k off of 10.2 s −1 . Together, our findings hint that previous experimental observations may have inadvertently captured the 3site state as an in vitro solution state, instead of the catalytic state alone, and provided a distinct departure from the conventional understanding of cytochrome P450.

Incorporating Prior Knowledge in the Seeds of Adaptive Sampling Molecular Dynamics Simulations of Ligand Transport in Enzymes with Buried Active Sites

“… 54 Here, we would like to note that the application of HMR might alter the time scales of the studied processes, as shown recently. 55 , 56 Hence, in studies aimed at measuring the absolute rates, unlike the primarily comparative work here, it might be advisable to avoid HMR.…”

“…Such a system was then processed with the tleap module of AMBERTools18, placing the presolvated proteins in the octahedral box of OPC water molecules at a distance of 10 Å and neutralizing them with counterions (Na + and Cl – ) at an ionic strength of 0.1 M. Finally, the hydrogen mass repartitioning (HMR) method was applied to produce topologies to enable a 4 fs time step . Here, we would like to note that the application of HMR might alter the time scales of the studied processes, as shown recently. , Hence, in studies aimed at measuring the absolute rates, unlike the primarily comparative work here, it might be advisable to avoid HMR.…”

Substrate Conformational Switch Enables the Stereoselective Dimerization in P450 NascB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

“…Despite the aforementioned mechanistic insights, the molecular mechanism of the P450-catalyzed biosynthesis of DKPs is not fully elucidated, especially regarding the protein environment effects in dictating the regio- and stereoselective C–N and C–C coupling. Extensive studies have shown that the protein environment is key to the activity and selectivity of enzyme catalysis, while the neglect of such an effect in the QM model calculations may give biased descriptions of the kinetic and thermodynamic properties of enzymatic processes. ,− In this study, we reexamined the molecular mechanism of the P450 NascB with the combined MD simulations , and quantum mechanical/molecular mechanical (QM/MM) calculations. − Particularly, we systematically explored all conceivable catalytic pathways originating from two distinct binding modes of Substrate 1. Our multiscale calculations reveal that pathway B, which involves a conformational movement of the substrate radical, is the most favorable reaction pathway.…”