into the DsrB1 subunit and vice-versa. This is the second residue in the heme road. The T351 residue in the DsrB subunit is aligned to N393 in the DsrA subunit in the sequence alignment, but structurally they are not in homologous positions (Figure S4). T351 is found at the N-terminal end of a helix in the central interface between heterotetramers in a T351B1-T351B2 interaction, which is the sole interaction at the central interface between heterodimers. The distance between the carbonyl oxygen atoms of the backbone of these two residues is 4.1 Å. This is the third residue of the heme road between the two functional siroheme moieties (Figure 5). Residues N180B1/2 and N393A2/1 are separated by only 10.1 Å, with two intervening aromatic residues, while a single aromatic residue separates N393A2/1 from T351B1/2 (Figure 5D). Thus while the heme road residues are not in direct contact, they are coupled by intervening aromatic side chains.
In support of evolutionary covariance of heme road residues, EVCoupling analysis of the MV2-Eury A subunit also indicated coupling between N369A and L204A, with equally probable residues involved in coupling being R337 and S167. These residues are the structural and sequence homologues of the four residues detected as monomeric FPECs by EVCoupling analysis of the A. fulgidus A subunit, three of which constitute the heme road. The third detected FPEC using the A. fulgidus B subunit sequence as bait, I350B-R197B (Table 1), implicates K240A and N392A as equally likely to be involved in a residue pair coupling. Residues N392A and I350B are adjacent to two residues in the heme road, N393A and T351B, providing support for their evolutionary coupling. Thus, T351B1-N393A (italicized and underlined in Table 1) are likely to correspond to actual co-varying residues. Given the relatively close proximity of N393B1 and N180B1, 10.1 Å, we hypothesize that these two residues may also be evolutionarily coupled. The aromatic residues linking the heme road residues, Y348B1, F394A2, in A. fulgidusare replaced in MV2-Eury by hydrophobic residues L334B1 and I360A2, and F317A1 is replaced by a proline, P166B1, from the B1 rather than A1 subunit.