Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

Abstract

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons : 1 dioxygen : 1 product. However, three alternate unproductive pathways exist where the intermediate iron-oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen-oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4 reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase.

Iron(IV)hydroxide p K a and the Role of Thiolate Ligation in C–H Bond Activation by Cytochrome P450

“…Under turnover conditions with substrates that fit poorly in the active site, these processes are known to lead to oxidase chemistry. (31–33) In rapid mixing experiments with m -CPBA, they can present a major obstacle to the isolation of P450-I. (9–11) As we will show, CYP158 allows us to use this facet of P450 chemistry to our advantage.…”

Characterization of the Active Site Properties of CYP4F12

“…Furthermore, the P450 catalytic cycle is known to include three nonproductive (uncoupling) pathways, of which only the formation of water from Compound I results in an NADPH to O 2 ratio of 2:1. The approximate twofold increase in the NADPH consumption rate that was observed for CYP4F12 and astemizole-d 3 with no increase in the formation of H 2 O 2 suggests that the enzymatic reaction may be branching to this uncoupling pathway and resulting in the formation of an additional water molecule from Compound I (Atkins and Sligar, 1988;Grinkova et al, 2013). Finally, to rationalize the previously discussed results using the active site geometries of CYP4F12 and CYP3A4, a homology model was designed for CYP4F12 and compared with an X-ray crystal structure model of CYP3A4.…”

Advances in the use of nanoscale bilayers to study membrane protein structure and function

“…This effect may underpin the recently reported increase 88 K. Malhotra and N.N. Alder in CYP3A4 coupling when reconstituted in the presence of anionic phospholipids (Grinkova, Denisov, McLean, & Sligar, 2013). As another experimental approach, neutron reflectometry was employed to monitor redox-dependent structural transitions of nanodisc-bound CPR aligned at the water-silica interface, suggesting that NADPH reduction promoted a compact conformation as a potential mechanism to prevent offpathway electron transfer (Wadsäter et al, 2012).…”