Sequences belonging to this clan are considered to be located on the inner membrane of mitochondria, as are the vertebrate members of this clan (CYP11, CYP24, CYP27). This clan has therefore the “mitochondrial” designation. This may be unfortunate and potentially confusing, because clan names refer to similarities in sequence, not subcellular localization, and because it is far from certain that each and all Mito clan P450s are actually mitochondrial.This clan probably should have been called the CYP11 clan (as the other clans are named after the first family of the clan).

The Mito clan is generally small, down to 4 sequences in the collembolan Sinella curviseta, but can be larger than the CYP2 or CYP4 clan as in the cat flea Ctenocephalides felis where Mito clan P450s comprise 30% of the CYPome (Feyereisen, 2022).

Strong evidence that house fly CYP12A1 is indeed a mitochondrial enzyme comes from its localization in mitochondria by immunogold histochemistry and absolute dependence on mitochondrial electron donors adrenodoxin reductase and adrenodoxin (Guzov et al., 1998). Such stringent criteria have not been applied to other members of this clan in arthropods, and assumption of mitochondrial localization is generally accepted, although Drosophila melanogaster CYP314A1, 302A1 and 315A1 tagged and expressed in S2 cells colocalize with a mitochondrial marker by confocal microscopy (Petryk et al., 2003).

In accordance with experimental evidence, the mitochondrial CYP12A1 is predicted to be targeted to mitochondria by DeepLoc, LocTree3 and SubCons. However, this is not the case for all Mito clan P450s. Of 306 Mito clan P450s in Dermauw et al., 2020, while 97.4% were predicted to be mitochondrial by LocTree3, only 81.4% and 72.9% were predicted to be mitochondrial by DeepLoc and SubCons respectively.

The case of CYP314A1 is of interest because it encodes an ecdysone 20-hydroxylase which is known to be found in both mitochondria and microsomes, depending on the species, the tissue and the developmental time (Lafont et al., 2012). The Bombyx mori 20-hydroxylase in eggs is microsomal and inhibited by (microsomal) NADPH cytochrome P450 reductase antibodies (Horike and Sonobe, 1999), and so is the larval cockroach enzyme (Halliday et al., 1986). It is not known whether these enzymes are encoded by CYP314A1, which is predicted to be located in the ER, not mitochondria by SubCons and DeepLoc in Bombyx mori and Locusta migratoria. While LocTree3 predicted all 38 CYP314A1 to be mitochondrial, this was only the case for 12 by DeepLoc and just 9 by SubCons.

The Anopheles funestus CYP314A1 shows 20 hydroxylase activity when coexpressed in E. coli with P450 reductase, suggesting that it is a microsomal enzyme (Ismail et al. ICE Helsinki 2022 presentation).

The “mitochondrial” in the name of this clan should therefore NOT be taken as definitive evidence of subcellular localization. Some P450s of this clan may be microsomal or have dual localizations.

Three widely distributed orthologs, CYP302A1, CYP314A1 and CYP315A1 are involved in ecdysteroid metabolism.

A number of P450 families from this clan, notably the CYP12 family in Diptera and the CYP333 family in Lepidoptera, contain many paralogous genes encoding typical xenobiotic metabolizing P450s (see Arthropod P450 functions ). Some of these genes are known to be linked to insecticide resistance.

CYP428A1 in Lepidoptera is an unusual member of the Mito clan, but predicted to have a mitochondrial presequence and to be destined to the inner mitochondrial membrane. Its sequence is highly conserved, but very distinctive from other mito clan P450s.

The consensus sequence around the Cys pocket motif has a three codon deletion, ASMPFG - - - x x CPxxG when compared with the consensus ASLPFGFGPRMCIGRR. Also, the CYP428A1 sequences have just one of the three positively charged residues known to favor binding of the adrenodoxin electron donor and have an unusual I helix, so they may use a different electron donor or may be independent of an external source of reducing equivalents (e.g. if acting on an endo-hydroperoxide).

CYP428A1 is not found in Trichoptera or in non-Ditrysian Lepidoptera, although genomic data in those groups are scant.