Cell Biochemistry and Biophysics

, Volume 63, Issue 1, pp 35–45 | Cite as

What is the Functional Role of N-terminal Transmembrane Helices in the Metabolism Mediated by Liver Microsomal Cytochrome P450 and its Reductase?

  • Daniel Andrew Gideon
  • Rashmi Kumari
  • Andrew M. Lynn
  • Kelath Murali Manoj
Original Paper

Abstract

We sought to clarify on the hitherto unresolved role of N-terminal transmembrane segments (TMS) of cytochrome P450 (CYP) and its’ reductase (CPR) in protein interaction/catalysis. TMS analyses show little evolutionary conservation in CYPs. The conserved CPR’s TMS poses limited scope for predictable/consistent hetero-recognition with the wide bevy of CYPs’ TMS, as evident from preliminary analyses and TMhit server predictions for inter-helical binding. Further, experimentations with four different CPR preparations (preps) and two liver microsomal CYPs (2C9 and 2E1) shows that the hydroxylated product formation rate is not quantitatively correlated to the extent of integrity of the CPR N-terms. Incorporation of cytochrome b5 in some reactions afforded similar rates while employing either fully intact or partially intact CPR. A survey of literature shows that liver microsomal CYPs function quite well even without the TMS or with significantly altered TMS. These observations negate the hypothesis that N-term TMS of CPR or CYP is obligatory for CYP–CPR interaction and catalysis. Also, in CYP2E1-mediated hydroxylation of para-nitrophenol, the extent of intactness or truncation did not significantly affect the CPR preps’ catalytic role at very low or high substrate concentrations. To interpret these results, we draw support from recently published research on reduced nicotinamide adenide dinucleotide phosphate oxidase (Takac et al., J Biol Chem, 286:13304–13313, 2011) and from our pertinent earlier works. We infer that CPR’ free TMS segment could alter the diffusible reactive oxygen species’ dynamics in the microenvironment, thereby altering the reaction outcome. Based on the evidence, we conclude that TMS merely facilitates “interaction/catalysis” by anchoring the CYP and CPR in the lipid interface.

Keywords

Helical interactions CYP Hetero-recognition Membrane anchoring Liver microsomes 

Abbreviations

CYP

Cytochrome P450

CPR

NADPH-cytochrome P450 reductase

NADPH

Reduced nicotinamide adenide dinucleotide phosphate

TMS

Transmembrane segment

TMH

Transmembrane helix

CO

Carbon monoxide

OD

Optical density

DLPC

Dilauryl phosphatidylcholine

HPLC

High performance liquid chromatography

pNC

Para-nitrocatechol

pNP

Para-nitrophenol

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Nox

NADPH oxidase

DROS

Diffusible reduced oxygen species

Supplementary material

12013_2012_9339_MOESM1_ESM.doc (1.4 mb)
Supplementary material 1 (DOC 1485 kb)

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Daniel Andrew Gideon
    • 1
  • Rashmi Kumari
    • 2
  • Andrew M. Lynn
    • 2
  • Kelath Murali Manoj
    • 1
  1. 1.Heme & Flavo Proteins Laboratory, Center for Biomedical ResearchVIT UniversityVelloreIndia
  2. 2.School of Computational and Integrative SciencesJawaharlal Nehru UniversityNew DelhiIndia

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