Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

Abstract

Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation. It has been hypothesized that the Pdx-induced shift toward the open state frees the essential Asp251 from salt bridges with Arg186 and Lys178 so that Asp251 can participate in a proton relay network required for O2 activation. This in part explains why P450cam has such a strict requirement for Pdx. One problem with this view is that looser substrate-protein interactions in the open state may not be compatible with the observed regio- and stereoselective hydroxylation. In the present study, molecular dynamics simulations show that Pdx binding favors a conformation that stabilizes the active site and decreases camphor mobility yet retains a partially open conformation compatible with the required proton relay network. The R186A mutant which frees Asp251 in the absence of Pdx retains good enzyme activity, and the crystal structure shows that product, 5-exo-hydroxycamphor, is bound. This indicates that rupture of the Asp251-Arg186 relaxes selectivity with respect to source of electrons and enables X-ray generated reducing equivalents to support substrate hydroxylation. These combined computational and experimental results are consistent with the proposed role of Pdx in assisting the release of Asp251 from ion pairs so that it can participate in proton-coupled electron transfer.

Effect of redox partner binding on CYP101D1 conformational dynamics

“…The analysis is similar to a recent study carried out with P450cam [9]. The percent of 5000 snapshots over a 100ns simulation where the C5-Fe distance is ≤ 4.5Å (Figure 3) relative to other camphor carbon atoms is presented in Table 2.…”

“…The binding of camphor to CYP101D1 was about 10 fold weaker compared to P450cam (K D ~1.2 μM). Table 1 provides a comparison with the ITC results obtained with P450cam [9]. In both cases ΔH is positive so the binding of camphor is entropically driven.…”

“…This enables x-ray induced reducing equivalents to drive substrate hydroxylation in crystals. Further evidence along these lines is that when the Asp-Arg ion pair is disrupted in P450cam, the enzyme retains substantial activity and product now forms in the x-ray beam similar to CYP101D1 [9]. These comparative studies between P450cam and CYP101D1 have provided important insights on how redox partner binding is coupled to the O 2 activation machinery enabling a fairly detailed mechanistic picture to emerge.…”

“…The methods used for molecular dynamics simulations were the same as previously published for P450cam [9]. The starting structure was the ferric camphor bound structure (4C9K).…”

“…This means that the open form is active in substrate hydroxylation and that the substrate must be held in the correct position for regio- and stereo-selective hydroxylation for x-ray driven reduction and O 2 activation. Recent computational studies have shown that Pdx stabilizes the active site and actually favors conformational states between the extremes of the open and closed forms [9]. This effector role of Pdx has been attributed to the requirement that an opening of the active site is necessary to arm the proton relay network essential for O 2 activation.…”