Efficient Bioelectronic Actuation of the Natural Catalytic Pathway of Human Metabolic Cytochrome P450s

Abstract

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo-and regioselective synthesis, and biosensors. We describe herein nm-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations support a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.

Electrochemistry-mass spectrometry to study reactive drug metabolites and CYP450 simulations

“…There are a few strategies describing the modification of several electrode materials with CYP450 [64][65][66], but some of these are often confronted by the method used for the electrochemical regeneration of the enzymatically active form of CYP450. Another advantage of modified CYP450-electrodes is that they can be appropriate for the routine generation of various metabolites [67,68]. Both mediated and unmediated processes have been reported, but the direct electrochemical regeneration of the active form of CYP450 still remains inefficient at this stage and displays low conversion rates.…”

Thin multicomponent films for functional enzyme devices and bioreactor particles

“…We made LbL films using CPR microsomes and pure cyt P450s that achieved a large ratio of cyt P450 to CPR (Figure 7), as exists in the human liver, to mimics the natural biocatalytic pathway of cyt P450s. vii Enzyme activation begins with initial electron injection from the electrode to CPR, which then transfers electrons to cyt P450 to combine with oxygen. viii When cyt P450s were chemically reduced to the Fe II form and reacted with CO, cyt P450 Fe II -CO spectral bands at 450 nm were found, confirming that native enzymes were present in the films.…”

“…vi This research culminated in our development with Sadagopan Krishnan of the first cyt P450 films to enable electrochemical activation of the natural catalytic cycle of this important class of oxidative metabolic enzymes. vii,viii …”

Continuous monitoring of Naproxen by a cytochrome P450-based electrochemical sensor

“…In several reports, CYP1A2 peaks were detected at À 380 mV (Krishnan et al, 2011b), À 265 mV (Antonini et al, 2003;Paternolli et al, 2004), À280 mV (Estavillo et al, 2003), and À 437 mV (Shumyantseva et al, 2007). Even more complex is the situation when microsomal CYPs are immobilized on electrodes.…”