## 3 Results and Discussion
### In Silico Mutagenesis and Biochemical Characterization of Putative Heme Binding Sites  
We used the Rosetta score function to estimate free energy changes following the in silico mutagenesis of the two putative heme-binding sites (H150 and H266) in the maize Z-ISO AlphaFold model. Unlike AlphaFold, which scores protein-folding accuracy based on an internal confidence probability derived from the machine learning procedure, Rosetta uses a scoring system related to thermodynamic free-energy changes (known as Rosetta energy units, REU) based on calculations of physiologically relevant parameters [39]. The H150A substitution resulted in a small increase in relative free energy compared to WT (\(-1104\) and \(-1109\) REU, respectively), possibly indicating that this substitution alters protein folding, which may explain the decreased and altered heme binding by this mutant observed previously [5]. No inter-domain interactions were identified at the H266 site and the H266A substitution resulted in only a marginal free energy decrease relative to WT (\(-1110\) and \(1109\) REU, respectively), likely due to the preference for hydrophobic residue packing in the absence of a cofactor.  
We further investigated the prospective heme binding sites by performing in silico mutagenesis around the H150 and H266 residues and estimating the free-energy changes using Rosetta. We sought to change the chemical nature of residues (e.g., His to Ala) or introduce a steric clash (e.g., His to Trp) to block the binding cavity. We also aimed to maintain the protein structure using mutations that would have a minimal effect on folding (i.e., minor free-energy changes). For the H266 site, H354 (approximately axial to H266; Figure 6D) was mutated to Ala or Trp, with relatively minor free-energy changes (+1 and +5 REU; Figure S8C). For the H150 site, we analyzed mutations to three proximal residues (Figure 6E), R170, Q255, and R296, changing each to Ala, Phe, or Trp. The Ala and Trp substitution in R170 yielded the most minimal free-energy changes of the three residues (Figure S8C). Thus, the H354A, H354W, R170A, and R170W variants were selected for production in _E. coli_. The membrane extracts and crude protein preparations were visible colored for all four of these protein variants, suggesting that they all bound at least some heme. A comparison of the oxidized and reduced spectrum of the R170A and R170W crude protein extracts showed a typical shift upon the addition of sodium dithionite, suggesting that neither of these substitutions prevent heme binding to Z-ISO (Figure 7A,B). Although pigmentation was observed in the H354A and H354W mutants, no significant change was observed between the oxidized and reduced spectra (Figure 7C,D) [5]. This suggests that heme may be weakly bound by the remaining histidine ligand (H266), but without the requisite sixth coordination bond, the addition of sodium dithionite is insufficient to reduce the ferric center, and hence no spectral changes are observed. In combination with the above modeling, these preliminary results suggest that H266 and H354 are likely to coordinate heme B, while H150 may instead have a structural or catalytic role.