Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis

Abstract

As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we Abbreviations: P450 BM3, cytochrome P450 monooxygenase from B. megaterium; P450 cam, cytochrome P450 monooxygenase from P. putida; WT, wild type; k cat , R-H, turnover rate constant with respect to hydroxylation; K m , R-H, Michaelis constant with respect to hydroxylation; k cat , leakage, turnover rate constant with respect to leakage; K m , leakage, Michaelis constant with respect to leakage; K d , dissociation equilibrium constant; k on , association rate constant; k off , dissociation rate constant; T 50 , incubation temperature at which half of protein becomes denatured after 10 min; NADPH, nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; H 2 O 2 , hydrogen peroxide; O 2 2 , superoxide; IPTG, isopropyl b-d-1-thiogalactopyranoside; KPi, potassium phosphate; HRP, horseradish peroxidase; ABTS, 2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); CO, carbon monoxide; Tris, tris(hydroxyl)aminomethane; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2015 The Protein Society investigated were far more deleterious than other mutations that have been used to change substrate specificity.

BEX1 Is Differentially Expressed in Aldosterone-Producing Adenomas and Protects Human Adrenocortical Cells From Ferroptosis

“…[33][34][35] Adrenocortical cells are especially sensitive to ferroptosis, 36,37 an observation likely related to the vulnerability of steroidogenic tissues to redox imbalance caused by electron leakage by cytochrome P450 enzymes and reactive oxygen species generation. [38][39][40][41] Herein, we report an inverse correlation of BEX1 gene expression with APA diameter and an upregulation of BEX1 transcription in micro-APAs, but not macro-APAs, compared with paired adjacent adrenal cortex. The differential expression levels of BEX1 in different adrenal tissue samples may reflect different levels of steroidogenesis and production of reactive oxygen species.…”

“…33–35 Adrenocortical cells are especially sensitive to ferroptosis, 36,37 an observation likely related to the vulnerability of steroidogenic tissues to redox imbalance caused by electron leakage by cytochrome P450 enzymes and reactive oxygen species generation. 38–41…”

Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

“…A low CE value indicates futile NADPH usage, resulting in the formation of reactive species during the catalytic cycle (Supplementary Fig. 1 ) that can inactivate the enzyme 47 . Low CE values of 15–30% were found for all mutants, except for -II and III that display higher values of 37% (Fig.…”

The Oxygen Dilemma: A Severe Challenge for the Application of Monooxygenases?

“…However, it should also be mentioned here that this approach does not always lead to improved self‐sufficient enzymes. Even in P450 BM3 the uncoupling can be as high as 60 % or even more …”