The b5 cores of Ncb5or and the rice RLF protein have identical folds.
The rice RLF construct crystallized readily as a non-crystallographic dimer and could be modeled from Phe111-Glu218 in subunit A and Lys113-Phe213 in subunit B. The fold comprises six α-helices and four β-sheets (Figure 4A-B ) that are arranged as follows: α1 (Ser116-Thr126), β1 (Arg140-Ile142), α2 (Leu144-Lys148), β2 (Trp157-Leu160), β3 (Arg163-Asn166), α3 (Ala168-Phe173), α4 (Val178-Met182), α5 (Thr190-His197), α6 (Phe202-Leu205), β4 (Leu210-Leu214). Note that the numbering is based on the NCBI reference sequence XP_015647767.1. The b5 core of RLF is identical to that of the Ncb5or b5 domain, with superposition yielding an RMSD deviation of 1.01 Å (80 residues) between Cα atoms (Figure 5 ).
The heme molecules are positioned within a cleft formed by helices α3-α6. It is important to note that the corresponding helices in the published structure of the Ncb5or b5 domain, and in structures of single domain b5 family members, are numbered α2-α5. In the comparison of the b5 and N/b5 structures we will refer to the relevant helices exclusively as α3-α6. Like all cytochrome b5 family members, the heme iron is coordinated by the side chains of two histidine residues (His174 and His197). His174 (His89 in Ncb5or) is located in the loop separating α3 and α4, while His197 (His112 in Ncb5or) constitutes the C-terminal residue in helix α5. As observed for His89 and His112 in the crystal structure of the Ncb5or b5 domain, the imidazolyl moieties of His174 and His197 in the rice RLF structure are nearly orthogonal to one another (Figure 6B ). The angle between the mean planes defined by the indole rings is 77.6o (subunit A) and 77.2o (subunit B) as calculated using Mercury.49 The corresponding angles for His89 and His112 in the two subunits of the Ncb5or b5 domain structure (3LF5) are 83.2° and 81.3°.8 This structural feature distinguishes these proteins (and likely IRC21 as well), from the better known microsomal and mitochondrial isoforms of cytochrome b5, in which angles between the His ligand side chains are closer to 20°.8
We reported that the b5 core of Ncb5or differs from the better known microsomal and mitochondrial isoforms of cytochrome b5 in having a strictly conserved tryptophan residue (Trp114) at the mouth of the heme binding pocket, two residues removed from heme ligand His112. The intervening residue, Arg113, is invariant among mammalian Ncb5or isoforms. In the crystal structure of the b5 domain of Ncb5or the side chain of Trp114 is located on the protein exterior and is substantially solvent-exposed (Figure 6A ), with its only inter-protein interactions involving the side chain of adjacent residue Arg113. The rice RLF residues corresponding to the Ncb5or His112ArgTrp114 sequence (His197, Ala198, and Trp199) are strictly conserved among known members of this protein family. In the RLF crystal structure the Trp199 side chain projects into solvent, but as shown in Figure 6A its side chain conformation differs from that of Trp114 in the Ncb5or b5 domain structure, enabling hydrogen bonding between its Nε-H and one of the heme propionate groups. Manually changing the side chain torsional angles of Trp114 in the Ncb5or b5 domain crystal structure (χº = -53.9; χ2 = 115.7º) to match those of Trp199 in the new rice RLF structure (χº = 58.9; χ2 = 85.4º), using PyMol, showed that Trp114 could form an analogous hydrogen bond with heme without introducing unfavorable steric interactions. Given that this solvent exposed Trp residue is strictly conserved among all known Ncb5or and RLF proteins (and in IRC21 proteins as well), its ability to form a hydrogen bond with heme suggests that it serves an essential functional role.
Yet another distinguishing feature shared by the b5 cores of Ncb5or and rice RLF is an irregular helix (α6) in the heme-binding pocket, featuring a central kink that leaves Leu205 (Met120 in Ncb5or) without an intra-helical hydrogen bond. As will be noted in the following section, this kink plays an important role in interactions with the N-terminal region.
While carrying out the studies reported herein, we became aware of reports of b5 family members in Giardia and some other parasitic organisms with strong homology to the b5 domains of Ncb5or, RLF and IRC21 proteins. As shown by some representative examples inFigure S5 , these proteins feature an N-terminal region, albeit generally shorter than those in Ncb5or, RLF and IRC21 and without a Trp residue analogous to Trp37 in Ncb5or.50 Many of them have also maintained a surface tryptophan residue analogous to Trp114 of Ncb5or. The Giardia proteins were shown to exhibit redox potentials51 similar to those determined for Ncb5or,2 which are considerably more negative than for microsomal b5s. TheGiardia proteins and Ncb5or also share EPR spectroscopic signatures that are characteristic of orthogonal His ligands.51 It is reasonable to conclude that these proteins have evolved from the common N/b5 ancestor of Ncb5or, RLF, and IRC21, perhaps with divergent functions.50, 51 This notion is supported by the observation of cytochrome b5 proteins in protists (Trypanosomaand Dictyostelium) that share the same b5 core50 and the N-terminal motif as that in Ncb5or, including the Trp114 and Trp37 residues, respectively (Figure S5 ).