Methionine coordination to the heme iron and its displacement by nitric oxide in the CcoP subunit of Pseudomonas aeruginosa cytochrome cbb3 oxidase.

Ligand substitution is common in signaling mechanisms of heme proteins. Methionine at the heme iron can get displaced by both endogenous and exogeneous ligands, including NO. The latter is particularly relevant in low-O2 environments and affects properties of metalloproteins in electron-transfer (ET) chains. Inspection of the recent cryo-EM structures of cytochrome (cyt) cbb3 oxidases from Pseudomonas aeruginosa (Pa) suggested that FeS(Met) distances in the CcoP subunit are too long for Met ligation to the heme iron, yet this ligation is critical for high reduction potentials to support downhill ET to this terminal oxidase. Using spectroscopic and electrochemical characterization, we show that the two hemes of the CcoP subunit of Pa cbb3-2 oxidase (CcoP2) are Met-ligated. Two variants of Pa CcoP2 were examined: wild-type (WT, referred here as IMA) and the I185A/A187P mutant (referred as AMP). Thermal denaturation and electrochemistry experiments show that the IMA to AMP substitution increases the protein global stability by ∼5 kcal/mol, in both ferric and ferrous forms. Molecular dynamics (MD) simulations suggest that mutational perturbations near the Met ligand propagate to interfacial loops of the two domains. As with horse heart cyt c, NO binds to ferric Pa CcoP2 proteins, but this binding is less thermodynamically favored, even less so in the AMP variant. MD simulations illustrate the preference for the swapped architecture in CcoP proteins and we propose functional advantages of this arrangement for ET proteins.