## 3 Results and Discussion
### Modelling of Z-ISO for In Silico Structure-Function Analysis  
As a structure of Z-ISO remains elusive, we used a combination of AlphaFold [37], Autodock [40], and Rosetta [47; 48] to probe the folding homology and structure-function relationships in Slr1599 and the two best-studied plant Z-ISO homologs in silico. While these approaches provide useful information, we stress that the AlphaFold structures,free-energy estimates, and cofactor docking are all computational models and should not be considered replacements for experimentally determined structures, and we note the recent publication of a genomics-based approach to identify Z-ISO orthologs suitable for structural studies [49]. Model structures derived from AlphaFold simulations are available for the maize and _Arabidopsis_ Z-ISO enzymes from UniProt (identififiers B4 FHU1 and Q9 SAC0, respectively) and the Slr1599 model was generated using AlphaFold v2.1.2 (see Section 2.6 for details); all the models have high pLDDT confidence scores and the structural coordinate files and per-residue model confidence values for Slr1599 are available for download in the Supplementary Information. The superimposition of the model structures with the transit signal peptides removed for the plant enzymes shows a very high homology (RMSD 0.4-0.9 A; Figure 6A). In agreement with the previous predictions, the plant enzymes have seven TMHs and Slr1599 has six, with N- and C-termini on opposing sides of the membrane for the plant Z-ISOs and the same (lumen) side of the membrane for the _Synechocystis_ enzyme. Two histidine residues, H150 and H266, and a cysteine, C263, have previously been implicated in heme binding/Z-ISO activity in work with the maize enzyme and these residues are conserved in Z-ISO homologs (Figure 2 and Figure S3). Mutagenesis studies showed that H150 and H266 are the only histidine residues required for function in maize Z-ISO [5] and we found the equivalent H29 and H145 residues are important for activity in Slr1599 (Figure 5). Based on homology modelling with maize Z-ISO, [PERSON] et al proposed that H150 acts as the proximal ligand to the heme cofactor and H266 and C263 act as alternate distal heme ligands in a redox-regulated ligand-switching mechanism that activates/inactivated the enzyme [5]. However, the AlphaFold models predict that H150 and H266 are too far apart in the structures for bis-His co  
Figure 6: AlphaFold models of Z-ISO. (**A**) Backbone structures of the _Synechocystis_ (green), _Arabidopsis_ (cyan), and maize (magenta) Z-ISO enzymes. (**B**) Positions of the two putative heme (HEM) binding sites in the maize enzyme, with H266 (cyan) and H150 (yellow) acting as heme ligands. (**C**) Density for the lowest-energy mode identified for 9,15,9\({}^{\prime}\)-tri-cis-\(\zeta\)-carotene (purple) binding to maize Z-ISO. (**D,E**) Expanded views of the putative H266 (panel (**D**)) and H150 (panel (**E**)) binding sites. therefore used Autodock Vina to dock heme B into the maize and _Arabidopsis_ Z-ISO models to investigate possible alternatives for heme ligation. First, a very broad search area (125,000 A\({}^{3}\)) was used for both structures (see Section 2.6 for details). In _Arabidopsis_, four potential heme binding sites were predicted, two of which were of particular interest (Figure S4); one site is oriented near H150 and another in close proximity to H266 (maize enzyme numbering). The H266 site contains a second possible axial ligand to coordinate the heme Fe-center, H354, and is proximal to the suggested alternative heme-coordinating residue, C263 [5], however we note that H354 was not required for Z-ISO function in previous studies [5; 6]. Conversely, in the other site there is no residue to donate the requisite sixth coordinate bond to the Fe center, suggesting that H150 is involved in inter-domain H-bonding and has a structural role rather than facilitating cofactor binding (Figure S8B).