Proton Relay Network in the Bacterial P450s: CYP101A1 and CYP101D1

Abstract

Cytochrome P450s are among nature’s most powerful catalysts. Their ability to activate molecular dioxygen to form high-valent ferryl intermediates (Compounds I and II) enables a wide array of chemistries ranging from simple epoxidations to more complicated C–H bond oxidations. Oxygen activation is achieved by reduction of the ferrous dioxygen complex, which requires the transfer of an electron from a redox partner and subsequent double protonation to yield a water molecule and a ferryl porphyrin π-cation radical (Compound I). Previous studies of the CYP101 family of cytochrome P450s demonstrated the importance of the conserved active site Asp25X residue in this protonation event, although its precise role is yet to be unraveled. To further explore the origin of protons in oxygen activation, we analyzed the effects of an Asp to Glu mutation at the 25X position in P450cam and in CYP101D1. This mutation inactivates P450cam but not CYP101D1. A series of mutagenic, crystallographic, kinetic, and molecular dynamics studies indicate that this mutation locks P450cam into a closed, inactive conformation. In CYP101D1, the D259E mutant changes the rate-limiting step to reduction of the P450-oxy complex, thus opening a window into the critical proton-coupled electron transfer step in P450 catalysis.

Active Site Hydrogen Bonding Induced in Cytochrome P450cam by Effector Putidaredoxin

“…9 The establishment of such an intraprotein proton relay network would enable proton-coupled electron transfer to efficiently couple the electrons into product formation. 17 Consistent with this model, the IR spectroscopy indicates that Pdx binding to P450cam induces ∼35% population strongly hydrogen bonded to an active site water molecule. This interaction is likely to contribute to the establishment of the proton delivery network that underlies the mechanism of Pdx's effector role.…”

“…9,13−15 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 2). 16,17 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. 18,19 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 1).…”

“…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.…”

“…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 .…”

An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network

“…While rational protein design has been extremely successful, it has largely focused on the design of interactions directly with the substrate or cofactor. On the contrary, extended H-bond networks, particularly those mediated by water molecules, are well-known in natural enzymes; − however, reports of rationally incorporating such water-mediated interactions have been limited. − Building on our previous results, we herein report incorporation of a Glu residue, which is known to be important in both HCOs and bd oxidases, into Cu B Mb and F33Y-Cu B Mb through rational extension of the water-mediated H-bond network in its active site and show that the introduced Glu plays a key role in eliminating ROS formation. In addition, we have obtained insights into the role of Glu using high-resolution crystal structures and EPR of cryoreduced oxy-I107E-Cu B Mb.…”