Evolution of the cytochrome bd oxygen reductase superfamily and the function of CydAA' in Archaea

Abstract

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify three families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA' cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA' from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA' is the first isoform of cytbd containing only b-type hemes shown to be active when isolated from membranes, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

Fig. 1. Families within the cytochrome bd-type oxygen reductase superfamily.

The cytochrome-bd type oxygen reductase superfamily is divided into three families—qOR, OR-C and OR-N, primarily defined by the presence of the quinol binding site in the first, the presence of heme c binding site in the second and the abundance of OR-N enzymes in Nitrospirota. The above schematic represents the various subfamilies within each family that are defined by the phylogenetic clustering shown in Supplementary Fig. S1. The operon context and putative complex arrangement of each CydA-containing enzyme is also shown with a reference protein accession number and source microorganism. The potential gene duplication events are highlighted in yellow. A legend is also provided to mark the related conserved domains in the same colors and redox co-factors such as hemes and iron-sulfur clusters.

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.

Fig. 3. Distribution of cytochrome bd-type oxygen reductases in Archaea.

Concatenated gene alignments were made from the archaeal genomes in GTDB using Anvi’o. A phylogenetic tree was made from the concatenated gene alignments using FastTree. All CydA sequences were extracted from GTDB genomes using BLAST with an e-value of 1e⁻¹. The sequences were then filtered to remove CydA sequences without characteristics of the quinol binding site and then classified using a Hidden Markov Model (HMM)-based classifier trained to identify the families—qOR1, qOR2, qOR3 and qOR4a. CydA sequences from each family were then mapped back to each species, and visualized along with the species tree on the iTOL server. Most phyla of the domain Archaea were distinguished by color and a few classes of the phylum Crenarchaeota were labelled to emphasize the presence of CydAA’. It is clear that CydAA’ is almost exclusive to the order Thermoproteales and Desulfurococcales.

Fig. 4. Biochemical characteristics of CydAA’ from Caldivirga maquilingensis.

A. and B. UV-visible spectra of cytochrome bd-type oxygen reductase purified from Escherichia coli and Caldivirga maquilingensis, respectively. C. Pyridine hemochrome spectra of CydAA’ from Caldivirga maquilingensis revealing the absence of heme d in the partially purified enzyme. D. Oxygen reductase activity of CydAA’ from C. maquilingensis shows that it is highly active and cyanide insensitive. It is sensitive to Aurachin C1-10, a quinol binding site inhibitor that also inhibits E. coli cytochrome bd.