With this goal in mind, we synthesized the bis-meso-substituted Fe(III)-porphyrin 3, prior to the introduction of the two anionic biotinylated anchors. To synthesize the N-bocprotected bis-meso-substituted porphyrin 2, we condensed di(1H-pyrrol-2-yl)methane 1 with N-boc-2-aminoacetaldehyde, and then oxidized the tetrapyrrole with DDQ to afford the aromatic porphyrin 2. Next, the porphyrin 2 was metalated with FeBr2 under anaerobic conditions, using 2,6-lutidine as a base. The formed Fe(II)-porphyrin when exposed to air, gets oxidized to form Fe(III)-porphyrin 3. Finally, the biotin-L-cysteic acid 4a was coupled to the deprotected metalated porphyrin 4 using HATU and NEt3 as reagents to afford the cofactor (Biot2 ─ FePP, Scheme 2, see SI for full experimental details). The anchoring of two Biot2 ─ FePP within the homotetrameric Sav S112A K121A (Sav AA, hereafter) mutant was confirmed by a peak at 67762 m/z in native HRMS experiments: the calculated peak for Biot2 ─ FePP • Sav AA is 67761 m/z, Figure . We also observed a minor peak in the deconvoluted mass spectrum that suggests the formation of a tetrameric protein bound to three Biot2 ─ FePP cofactors. We hypothesize that if the second biotin of the cofactor points outward of the binding pocket, two Sav monomers, facing each other, may accommodate two Biot2 ─ FePP cofactors instead of one.