Discussion

Overall structure comparison

Above are ribbon with semi-transparent surface structures of some  members of HCO superfamily: (A) qNOR, (B) cNOR, (C), cbb3 oxidase, (D) ba3 oxidase [B-type COX]. Red box indicates structural homology of TMH I qNOR. Hydrophilic domain of qNOR is also structurally conserved in other proteins, despite it not having any heme group. cbb3 oxidase CcoO subunit and qNOR hydrophilic domain have an extra helix from those in NorC subunit, although the position of the extra helix varies

Below is the table describing briefly the general similarities and differences of the four HCO proteins: 

Feature	qNOR	cNOR	cbb3 oxidase	ba3 oxidase
Calcium ion	✓	✓	✓	Two charged Arg
NO reduction activity	✓	✓	✓ (partially)	x
Hydrophilic domain folding	cyt-c (ligand not present)	cyt-c	cyt-c	cupredoxin
Proton transfer mechanism	water channel	cytoplasmic proton transfer	K-pathway (reducing O2)	K- and D-pathways
Electron donor	menaquinol	cytochrome-c551	cytochrome-c551	cytochrome-c552
Non-heme metal	Fe	Fe	Cu	Cu

Binuclear centre

(A) qNOR, (B) cNOR, (C) cbb3 oxidase, and (D) ba3 oxidase (Type B COX)
Residues around this centre confers selectivity to the reactant, i.e. either NO or O2

Three histidine ligands of qNOR and cNOR non-heme metal have similar configuration, while COX ligands  (i.e. cbb3 and ba3 oxidases) favour a more planar geometry present in copper coordination due to histidine-tyrosine covalent bond.

At least a substitution of Gly553  in qNOR to tyrosine is required to confer a COX-like binuclear centre, whereas Glu512 substitution to tyrosine will yield a cbb3-oxidase-like centre.
Significant homology between four proteins is apparent from the similar structural arrangements with generally conserved residues around the binuclear centre.

Electron transfer pathway

As qNOR has menaquinol as its reductant, the cytochrome-c fold in the hydrophilic domain seems vestigial, athough the general conformation is retained through the presence of bulky hydrophobic groups (Tyr84, Tyr98, Tyr170, Tyr171, Phe175, Leu183 and Phe214).

NOR and cbb3 oxidase have a conserved Ca between the heme b and b3. Only A/B-type COXs do not have a conserved Ca ligand, as two charged Arg residues are present instead in bridging the two hemes.

Proton transfer pathway

Although very related, qNOR purportedly doesn’t have the same proton transfer pathway as cNOR.

Conserved Asp198 in cNOR (B) is substituted with hydrophobic residue (Ala:53%, Val:12%, Ile:14%, Thr:10%, Met:7%) in qNOR (A). Hence, instead of proton coming from the extracellular side like cNOR, the qNOR water channel connected to the cytoplasmic side provides the proton source.

(A) qNOR, (B) cNOR, (C) cbb3 oxidase
In cNOR, the supposed water channel is blocked by Ile244 and Phe290 in place of Gln545 and Glu59 found in qNOR. Despite that, cNOR has conserved Glu211, Glu215, Glu280, and Tyr356 residues below the binuclear centre.

K-pathway in cbb3 oxidase structurally somewhat homologous with both cNOR choked water channel and qNOR water channel, with some residues overlapping especially the ones near the binuclear centre (Tyr317; Thr215/Ser283 analogous to Glu516/Glu215 in qNOR/cNOR respectively), albeit having a different entry side at cytoplasmic side.

Below is a schematic diagram to illustrate the differences in proton transfer between cNOR, qNOR, and COX.

Evolutionary relationship

Structural comparison between cyt-cs and cyt-c like domains of respiration enzymes
	Cytochrome C551  82 AA	Cytochrome C 116 AA	NorC subunit of cNOR 142   AA	Hydrophilic region of qNOR	CcoO subunit of cbb3 oxidase 197 AA
Cyt-c551 from P. aeruginosa (PDB 351C)49 82 AA	-	76 AA	82 AA	77 AA	No substantial homologya
		2.29 A	3.53A	2.30A	b
		36.20%	27.90%	10.60%	c
Cyt-c from Rhodothermus merinus (PDB 3CP5)50  116 AA		-	88AA	No substantial homology	59 AAa
			3.58 A		3.13 Ab
			29.70%		6.40%c
NorC subunit of cNOR from P. aeruginosa (PDB300R)22  142AA				134 AA	126 AAa
				2.78 A	5.02 Ab
				29.50%	26.80%c
N-terminal hydrophilic region of qNOR 225AA				-	No substantial homology
CcoO subunit of cbb3 oxidase from Pseudomonas stutzeri (PDB 3MK7)26 197AA					-

a	in number of aligned amino acids
b	r.m.s. deviation of positions of the aligned residues
c	Normalised distance score defined by MATRAS

Table above, reproduced from reference paper, can help us work out the evolutionary relationship. We can deduce that:

• Highlighted in yellow : 134 out of 142 amino acids homologous alignment between hydrophilic region of qNOR and NorC region of cNOR, with 2.78A deviation of positions and 29.5% normalised distance score. Suggests that they are relatively evolutionary linked.
• Highlighted in green : likewise, 126 out of 142 amino acids homologous alignment between CcoO subunit of cbb3 oxidase and NorC subunit of cNOR, with 5.02A deviation of positions and 26.8% normalised distance score. Also suggests that they are relatively evolutionary linked.
• No substantial homology between CcoO subunit of cbb3 oxidase and N-terminal hydrophilic region of qNOR. cNOR, qNOR and cbb3 are evolutionary related but qNOR and cbb3 are not directly related.

Proton exit pathway in cbb3 oxidase to periplasm also overlaps with the proposed proton transfer pathway in cNOR. Given that cbb3 oxidase has some NO reduction activity, interestingly it does so by utilising proton coming from its exit pathway instead, thus analogous to cNOR. Reaction intermediate of NO reduction may have a generally low proton affinity, which makes coupling with cytoplasmic proton pumping difficult. This implies that cbb3 oxidase may be the intermediate between cNOR and A/B type COX like ba3 oxidase.

It is deduced that through several mutations, qNOR water channel and cbb3 K-pathway may be formed from cNOR, indicating that cNOR is the intermediate between the two proteins.

COX and NOR though share a more distant relationship, as apart from having a cupredoxin fold and Arg residues instead of Ca, it also has a gate for proton pumping, although qNOR water channel presents an analogous structure to proton pumping pathway in A/B-type COX.

Our proposed evolutionary relationship between the four proteins are as such:

qNOR is thought to evolve first due to nitrogen rich in early atmosphere. COX might have evolved from cNOR-like protein as oxygen concentration increases over time, using it as the oxidant.
A qNOR-like protein that could reduce NO to both N2O and O2 has been discovered recently. Its structural analysis could help understand the evolutionary relationship better.

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