A second, smaller tunnel also reaches the active site and may serve as a pathway for water, but is too narrow to transport the retinoid reactants and products.

A major portion of RPE65's hydrophobic face, residues 109–126, forms an amphipathic alpha helix that likely contributes to the protein's membrane affinity.

The presence of this channel on the hydrophobic face combined with RPE65's demonstrated ability to absorb substrate direction from the lipid bilayer is consistent with RPE65 being partially embedded in the membrane.

[8] RPE65 has been isolated from a wide range of vertebrates including zebra fish, chicken, mice, frogs, and humans.

[13] The histidine residues of the beta-propeller structure and the bound iron(II) cofactor are 100% conserved across studied RPE65 orthologs and other members of the carotenoid oxygenase family.

This theory suggested that the reversible conversion of sRPE65 to mRPE65 by palmitoylation at Cys231, Cys329, and Cys330 played a role in regulating the retinoid cycle and endowing mRPE65 with its membrane affinity.

[9][17] The O-alkyl cleavage of the ester bond, assisted by an Fe(II) cofactor, creates a carbocation intermediate that is stabilized by the conjugated polyene chain.

After isomerization, a nucleophilic attack by water at C15 restores the conjugation of the polyene chain and completes the ester bond cleavage.

However, isotope labeling studies have demonstrated that the oxygen on the final 11-cis-retinol product of RPE65 originates from the solvent rather than the reacting ester, supporting the O-alkyl cleavage mechanism.

Not only is the polyenyl ester probably not electron-poor enough to allow this reaction, but the active site region is lacking cystine residues to act as the nucleophile.

[19] The vast majority of RPE65 mutations in patients with LCA2 and RP occur in the beta-propeller regime and are believed to inhibit proper protein folding and iron cofactor binding.

[13] Though complete loss of function is associated with diseases such as LCA and RP, partial inhibition of RPE65 has been proposed as a treatment for age-related macular degeneration (AMD).