The Second Protonation in the Bio-Catalytic Cycles of the Enzymes Cytochrome P450 and Superoxide Reductase

The enzymes Cytochrome P450 and Superoxide Reductase, which have a similar coordination center [FeN4S], begin their biochemical cycles similarly. They absorb an oxygen molecule, add two electrons, and link a hydrogen atom to the distal oxygen atom of the product obtained, creating the so-called Compound 0 in the case of the first enzyme. However, the bio-catalytic processes of these two enzymes continue in different ways. In the bio-catalytic cycle of Cytochrome P450, the enzyme binds another proton to the distal oxygen atom, producing a water molecule and Compound 1. In contrast, in the bio-catalytic cycle of the Superoxide Reductase, the enzyme binds a proton to the proximal oxygen atom, producing a hydrogen peroxide molecule, which later decomposes into oxygen and water. The MCSCF method in the CASSCF form was used to study the difference in Cytochrome P450 and Superoxide Reductase’s bio-catalytic cycles. The results of these enzymes’ hydroperoxo adduct models’ geometric optimization showed that, in fact, all their properties, including their spin states, the wave functions in their active zones, and the Fe-N, Fe-S, and Fe-O bond lengths, are different. The Fe-N, Fe-S, and Fe-O chemical bond lengths are much longer in the case of the second enzyme compared to the chemical bond lengths in the case of the first enzyme, reflecting a spin value equal to 5/2 in the second case and a spin value equal to 1/2 in the first. A decisive role in the difference in their bio-catalytic cycles is played by the fact that the first bonded hydrogen atom is linked to the distal oxygen atom in the side position in the case of Compound 0 and the up position in the case of the hydroperoxo adduct of the enzyme Superoxide Reductase, protecting the distal oxygen atom from possible interaction with the substrate. The second protonation to Compound 0 at the distal oxygen atom in the case of Cytochrome P450’s bio-catalytic cycle and the second protonation at the proximal oxygen atom in the case of the hydroperoxo adduct of Superoxide Reductase’s bio-catalytic cycle depend on the proton transfer through the Asp251 channel in the first case and on the transferal of H+ from the substrate to the water molecule and the proximal oxygen in the second case.