title: 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 investigated were far more deleterious than other mutations that have been used to change substrate specificity.
authors: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.
authors: Joseph B
authors: JB
authors: Lim
authors: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.
authors: Kimberly A
authors: KA
authors: Barker
authors: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.
authors: Kristen A
authors: KA
authors: Eller
authors: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.
authors: Linda
authors: L
authors: Jiang
authors: Department of Chemical Engineering, Polytechnic University of Puerto Rico, San Juan, 00918, Puerto Rico.
authors: Veronica
authors: V
authors: Molina
authors: Neuroscience Program, Wellesley College, Massachusetts, 02481.
authors: Jessica F
authors: JF
authors: Saifee
authors: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, 02139.
authors: Hadley D
authors: HD
authors: Sikes
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: cytochrome P450; hydrogen peroxide; leakage; reactive oxygen species; site-directed mutagenesis; spin state; thermostability