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RPE65 is an iron(II)-dependent isomerohydrolase in the retinoid visual cycle.
Gennadiy Moiseyev1, Yusuke Takahashi, Ying Chen
1Department of Cell Biology, Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
The Journal of Biological Chemistry
|December 2, 2005
Summary
The protein RPE65, crucial for vision, requires iron (Fe2+) to convert all-trans-retinyl ester to 11-cis-retinol. This study confirms RPE65 as an iron-dependent isomerohydrolase in the visual cycle.
Area of Science:
- Biochemistry
- Molecular Biology
- Vision Science
Background:
- The visual cycle involves the isomerization of all-trans-retinyl ester to 11-cis-retinol in the retinal pigment epithelium (RPE).
- Protein RPE65 has been identified as the key isomerohydrolase responsible for this critical reaction.
- The requirement of metal ions for RPE65 activity remained uninvestigated.
Purpose of the Study:
- To determine if metal ions are essential for the isomerohydrolase activity of RPE65.
- To identify the specific metal ion, if any, required for RPE65 function.
- To elucidate the role of RPE65 as an iron-dependent enzyme in the visual cycle.
Main Methods:
- Utilized metal chelators (2,2'-bipyridine, 1,10-phenanthroline) to inhibit RPE65 activity in bovine RPE microsomes and recombinant RPE65.
- Assessed isomerohydrolase activity by measuring the conversion of all-trans-[3H]retinol to 11-cis-[3H]retinol.
- Investigated the effect of various metal ions (FeSO4, CuSO4, ZnCl2, MgCl2) on restoring inhibited enzyme activity.
- Employed iron-staining methods and inductively coupled plasma mass spectrometry to detect and quantify iron in purified RPE65.
Main Results:
- Metal chelators significantly inhibited RPE65 isomerohydrolase activity in a dose-dependent manner, while lecithin-retinol acyltransferase (LRAT) activity remained unaffected.
- The inhibited isomerohydrolase activity was restored by ferrous sulfate (FeSO4), but not by other tested metal ions, indicating a requirement for Fe2+.
- Recombinant RPE65 activity was also inhibited by metal chelators and restored by FeSO4.
- Purified RPE65 was found to contain endogenous iron, with bovine RPE65 binding iron with a stoichiometry of 0.8 ± 0.1.
Conclusions:
- RPE65 functions as an iron-dependent isomerohydrolase.
- Iron (Fe2+) is essential for the catalytic activity of RPE65 in the visual cycle.
- These findings provide critical insight into the molecular mechanism of RPE65 and the visual cycle.