Updating the Paradigm: Redox Partner Binding and Conformational Dynamics in Cytochromes P450

Abstract

Metrics & More Article RecommendationsCONSPECTUS: This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O 2 O−O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe 2 S 2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam.P450cam is specific for its own Fe 2 S 2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/ allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam−Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O 2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O 2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surro...

Unravelling the role of transient redox partner complexes in P450 electron transfer mechanics

“…S4 ). This resembles the conformational rearrangements induced by putidaredoxin in the crystal structure of bacterial ( Pseudomonas putida ) P450cam 28 , 30 , 31 .…”

Cooperative Substrate Binding Controls Catalysis in Bacterial Cytochrome P450terp (CYP108A1)

“…This switch allows for the formation and participation of Asp251 in a proton relay network required for the controlled delivery of protons to the Fe−O 2 unit for the activation and subsequent heterolytic cleavage of the O−O bond. 18,19,76 P450terp contains an analogous aspartate (Asp270) on the I helix followed by a conserved threonine (Thr271) also critical for oxygen activation in P450cam (Thr 251). 77,78 Like Asp251 in P450cam, Asp270 in P450terp appears to be tied up in a similar but possibly weaker salt bridge with F-helix residue Gln185 as well as Lys419, which reaches over from the β5 sheet.…”

“…The freeing of Asp251 is accompanied by a change in the rotomer conformation to a position that faces the active site. This switch allows for the formation and participation of Asp251 in a proton relay network required for the controlled delivery of protons to the Fe–O 2 unit for the activation and subsequent heterolytic cleavage of the O–O bond. ,, P450terp contains an analogous aspartate (Asp270) on the I helix followed by a conserved threonine (Thr271) also critical for oxygen activation in P450cam (Thr 251). , Like Asp251 in P450cam, Asp270 in P450terp appears to be tied up in a similar but possibly weaker salt bridge with F-helix residue Gln185 as well as Lys419, which reaches over from the β5 sheet. Both interactions, however, engage the same carboxylate oxygen atom of Asp270 leaving the remaining side chain oxygen to participate in interactions with water molecules (Figure S4).…”

“…As more P450s were discovered and the number of detailed mechanistic studies expanded, it became clear that P450cam exhibits some additional unusual regulatory properties. The earliest unique feature of P450cam to be discovered was its strict requirement for its own redox partner, while many other P450s can be supported by foreign redox partners. − We now know that Pdx binding to P450cam results in a Pdx-specific induced conformational change that triggers the formation of a proton relay network required for O 2 activation. − More recently, P450cam has been found to have a second allosteric substrate binding site and undergoes complex and large structural changes in response to the binding of heme iron ligands. , …”

P450 electron transfer mechanics: unravelling the evolutionary role of transient redox protein complexes

“…3). This resembles the conformational rearrangements induced by putidaredoxin in the crystal structure of bacterial (Pseudomonas putida) P450cam 28,30,31 .…”