Abstract
Two key amino acids, Thr252 and Asp251, are known to be important for dioxygen activation by cytochrome P450cam. We have solved crystal structures of a critical intermediate, the ferrous dioxygen complex (Fe(II)-O2), of the wild-type P450cam and its mutants, D251N and T252A. The wild-type dioxygen complex structure is very much the same as reported previously (Schlichting, I., Berendzen, J., Chu, K., Stock, A. M., Maves, S. A., Benson, D. E., Sweet, R. M., Ringe, D., Petsko, G. A., and Sligar, S. G. (2000) Science 287, 1615-1622) with the exception of higher occupancy and a more ordered structure of the iron-linked dioxygen and two "catalytic" water molecules that form part of a proton relay system to the iron-linked dioxygen. Due to of the altered conformation of the I helix groove these two waters are missing in the D251N dioxygen complex which explains its lower catalytic activity and slower proton transfer to the dioxygen ligand. Similarly, the T252A mutation was expected to disrupt the active site solvent structure leading to hydrogen peroxide formation rather than substrate hydroxylation. Unexpectedly, however, the two "catalytic" waters are retained in the T252A mutant. Based on these findings, we propose that the Thr(252) accepts a hydrogen bond from the hydroperoxy (Fe(III)-OOH) intermediate that promotes the second protonation on the distal oxygen atom, leading to O-O bond cleavage and compound I formation.
Highlights
Two key amino acids, Thr252 and Asp251, are known to be important for dioxygen activation by cytochrome P450cam
Since temperature factors and occupancy are highly correlated, it appears that the method we used for preparing the oxy complex leads to higher occupancy and/or a more ordered structure. While both molecules in the asymmetric unit have oxygen bound with essentially no differences, we will focus on molecule B, since in the mutants the temperature factors for the dioxygen ligand are lower in molecule B leading to shaper electron density for both the ligand and surrounding residues and water molecules
Thr252 Ala (T252A) consumes NADH at about 85% WT rates and is highly uncoupled, since reducing equivalents and protons are funneled into the formation of hydrogen peroxide rather than camphor hydroxylation
Summary
Thr252 and Asp251, are known to be important for dioxygen activation by cytochrome P450cam. The two “catalytic” waters are retained in the T252A mutant Based on these findings, we propose that the Thr252 accepts a hydrogen bond from the hydroperoxy (Fe(III)OOH) intermediate that promotes the second protonation on the distal oxygen atom, leading to O–O bond cleavage and compound I formation. Detailed kinetic isotope effect and proton inventory studies with the Asp251 Asn mutant showed that the Asp251 mutation decelerates the electron transfer step and associated proton transfer events [16] These studies clearly implicate Thr252 and Asp251 as critical residues in the dioxygen activation process and especially in the proper delivery of protons to the iron-linked dioxygen. The crystal structures of the Thr252 Ala (T252A) [17] and Asp251 Asn (D251N) [16] mutants of P450cam in the ferric (Fe(III)) state provided the first hints as to how these mutations alter activity. There is a significant rearrangement of water structure that provides a continuous hydrogen-bonded link between dioxygen, water molecules, and
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