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Human coproporphyrinogen oxidase is not a metalloprotein
1Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-2605, USA. Dailey@bscr.uga.edu
The Journal of Biological Chemistry
|December 20, 1996
Summary
Coproporphyrinogen oxidase (CPO) is not a metalloenzyme. Human CPO activity does not require copper or iron, challenging previous assumptions about its function in heme biosynthesis.
Area of Science:
- Biochemistry
- Enzymology
- Heme Biosynthesis
Background:
- Coproporphyrinogen oxidase (CPO) is crucial for heme production.
- Previous studies suggested CPO requires copper for activity.
- This study investigates the metal requirements of human CPO.
Purpose of the Study:
- To determine if human CPO contains a metal center.
- To assess the in vitro effect of copper and iron on CPO activity.
- To investigate the impact of metal supplementation during protein expression.
Main Methods:
- Engineered human CPO cDNA for expression in E. coli with a His6 tag.
- Purified recombinant human CPO using nickel-nitrilotriacetic acid resin.
- Assessed CPO activity via a coupled fluorometric assay and metal analysis (UV-Vis, ICP-AES, EPR).
Main Results:
- Purified human CPO exhibited an apparent Km of 0.6 microM and Kcat of 16 min-1.
- Spectroscopic and ICP-AES analyses revealed no metal center in human CPO.
- In vitro assays showed no stimulation by copper or iron; metal supplementation during expression had no effect.
Conclusions:
- Human CPO is not a cuproenzyme or metalloenzyme.
- CPO activity is independent of copper and iron.
- This finding refutes previous hypotheses regarding CPO's metal cofactor requirements.