Mitochondrial Targeted Cytochrome P450 2E1 (P450 MT5) Contains an Intact N Terminus and Requires Mitochondrial Specific Electron Transfer Proteins for Activity

Marie-Anne Robin,Hindupur K Anandatheerthavarada,Ji-Kang Fang,Mare Cudic,Laszlo Otvos,Narayan G Avadhani

doi:10.1074/jbc.m100363200

Marie-Anne Robin, Hindupur K Anandatheerthavarada + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m100363200

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Abstract

Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.

Highlights

Cytochromes P450 (P450s)1 belong to a superfamily of heme proteins, which catalyze the oxidation of exogenous as well as endogenous compounds
Mass spectrometry analysis of tryptic fragments and immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart
We have used a combination of protein purification, enzyme reconstitution, and protein biochemistry for characterizing the molecular and enzymatic properties of rat liver mitochondrial P450 2E1

Summary

Introduction

Cytochromes P450 (P450s) belong to a superfamily of heme proteins, which catalyze the oxidation of exogenous as well as endogenous compounds. Microsomal P450 is co-translationally inserted in the ER membrane through a non-cleavable N-terminal hydrophobic signal sequence [6]. Mitochondrial P450 forms, such as P450scc (P450 11A1), P450 11␤ (P450 11B1), and P450c27 (P450 27A1) contain cleavable N-terminal amphipathic presequences that are important for the post-translational targeting of the precursor protein to mitochondria [7]. The mitochondrial targeted P450 forms were referred to as P450 MT2 and P450 MT4, respectively These studies described a new concept of chimeric N-terminal signal that was responsible for targeting both of these proteins to two distinct cytoplasmic organelles. P450 2E1 has been implicated to have important roles in human health, as its activity is affected by pathophysiological conditions such as diabetes, starvation, and obesity [12] It is readily induced by acute and chronic alcohol ingestion, and the enzyme is known to actively metabolize alcohol and acetaldehyde. The role of mitochondrial targeted P450 2E1 in oxidative stress remains unknown

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