Proton relay network in P450cam formed upon docking of putidaredoxin

Abstract

Cytochromes P450 are versatile heme‐based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O─O cleavage step remain partly elusive to date; effects observed in various enzyme mutants remain partly unexplained. We have carried out extended (to 1000 ns) MM‐MD and follow‐on quantum mechanics/molecular mechanics computations, both on the well‐studied FeOO state and on Cpd(0) (compound 0). Our simulations include (all camphor‐bound): (a) WT (wild type), FeOO state. (b) WT, Cpd(0). (c) Pdx (Putidaredoxin, redox partner of P450)‐docked‐WT, FeOO state. (d) Pdx‐docked WT, Cpd(0). (e) Pdx‐docked T252A mutant, Cpd(0). Among our key findings: (a) Effect of Pdx docking appears to go far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (b) Specific proton relay networks we identify are: FeOO(H)⋯T252⋯nH 2O⋯D251 in WT; FeOO(H)⋯nH 2O⋯D251 in T252A mutant; both occur with Pdx docking. (c) Direct interaction of D251 with –FeOOH is, respectively, rare/frequent in WT/T252A mutant. (d) In WT, T252 is in the proton relay network. (e) Positioning of camphor appears significant: when camphor is part of H‐bonding network, second protonation appears to be facilitated.

Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin

“…Efficient proton transfer minimizes uncoupling through shunt pathways, promoting hydroxylation of the substrate to the product. 20 Although Pdx binding now has been well linked to conformational changes in P450cam, and simulations support the resulting formation of a proton delivery network, 14,17 details about the mechanism of Pdx's effector role are still being uncovered. Toward elucidating why catalysis by P450cam requires Pdx, we applied IR spectroscopy to CN − -ligated P450cam and characterized the enzyme in the absence and presence of Pdx.…”

“…25 However, in the absence of Thr252, the crystal structure and MD simulations of the O 2 complex of T252A indicate that water molecules maintain the water network between the ligand and Asp251; thus, Thr252 does not appear critical to establishing the network. 14,19 IR spectroscopy of CN − -ligated P450cam provides evidence that complexation with Pdx induces a new population of the enzyme. To help uncover the nature of the induced population, we also evaluated the effect of osmotic stress, H 2 O/D 2 O exchange, and another mutation of the enzyme, L358P, intended to perturb the proximal thiolate heme ligation.…”

“…Interest in Pdx’s effector role has been reinvigorated in recent years, driven in part by acquisition of crystal structures of complexes of P450cam and Pdx. − Crystallographic, spectroscopic, and computational studies have established that complexation with Pdx induces substantial conformational changes throughout P450cam. − Early crystallographic evidence indicated that Pdx binding causes a conformational change from the “closed” state of camphor-bound enzyme toward the “open” state found in the absence of substrate . More recently, the crystal structure of the CN – -ligated complex with Pdx, double electron–electron resonance spectroscopy of spin labels, and molecular dynamics (MD) simulations point to P450cam adopting an intermediate state between the closed state and open states. ,− A key aspect of the proposed intermediate state is the establishment of a water network for proton delivery from the protein surface to the O 2 ligand (Figure ).…”

“…Interest in Pdx’s effector role has been reinvigorated in recent years, driven in part by acquisition of crystal structures of complexes of P450cam and Pdx. − Crystallographic, spectroscopic, and computational studies have established that complexation with Pdx induces substantial conformational changes throughout P450cam. − Early crystallographic evidence indicated that Pdx binding causes a conformational change from the “closed” state of camphor-bound enzyme toward the “open” state found in the absence of substrate . More recently, the crystal structure of the CN – -ligated complex with Pdx, double electron–electron resonance spectroscopy of spin labels, and molecular dynamics (MD) simulations point to P450cam adopting an intermediate state between the closed state and open states. ,− A key aspect of the proposed intermediate state is the establishment of a water network for proton delivery from the protein surface to the O 2 ligand (Figure ). , This network might involve two “catalytic” waters captured in the crystal structures of the O 2 and cyanide (CN – )-ligated P450cam in the absence of Pdx. , Establishment of such a proton delivery network in concert with reduction by Pdx would enable immediate protonation of the ferric-peroxo intermediate ( 5 ) to generate hydroperoxy intermediate ( 6 ), followed by protonation, oxygen cleavage, and release of water to generate intermediate ( 7 ), known as compound I (Figure ).…”

“…Efficient proton transfer minimizes uncoupling through shunt pathways, promoting hydroxylation of the substrate to the product . Although Pdx binding now has been well linked to conformational changes in P450cam, and simulations support the resulting formation of a proton delivery network, , details about the mechanism of Pdx’s effector role are still being uncovered.…”