Fig. 2. Phylogeny of quinol-oxidizing cytochrome bd-type oxygen reductases.

Protein sequences of cytbd subunit I, CydA were extracted from a taxonomically diverse set of genomes and metagenomes from IMG, filtered with UCLUST using a percentage identity cut-off of 0.6 and aligned using MUSCLE. The multiple sequence alignment, MSA4 was used to infer a phylogenetic tree using RAxML. The RAxML tree topology was similar to that inferred by IQ-tree. The CydA sequences that do not contain the quinol binding site, from the OR-C and OR-N subfamilies as well as qOR4b, were used as the outgroup. At least four monophyletic clades of typical CydA sequences that contain the O₂- and quinol binding site could be defined—qOR1, qOR2, qOR3 and qOR4a. The long branch within the qOR1 clade comprises a number of cytbd that are highly similar to enzymes from this clade but are missing the proton channel. Subunit I of CydAA’ is from the qOR4a-subfamily while subunit II or CydA’ is from the qOR4b-subfamily.