Can the Replacement of a Single Atom in the Enzyme Horseradish Peroxidase Convert It into a Monoxygenase? A Density Functional Study Sam P. de Visser* Contribution from the Manchester Interdisciplinary Biocenter and the School of Chemical Engineering and Analytical Science, The UniVersity of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom ReceiVed: August 31, 2006; In Final Form: September 1, 2006 Density functional calculations on horseradish peroxidase mutants are presented, whereby one or two of the nitrogen atoms of the axial histidine ligand have been replaced by phosphorus atoms. Our calculations show that phosphorus entices a push effect on the oxoiron group, whereas a histidine side chain withdraws electrons. As a result, we predict that a phosphorus-substituted histidine ligand will convert the active form of a peroxidase into a monoxygenase. This subsitution may be useful for the bioengineering of commercially exploitable enzymes. Peroxidases are heme enzymes that catalyze the detoxification of hydrogen peroxide to water and molecular oxygen. 1 The active species (Compound I, CpdI) of heme peroxidases, such as horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP), contains an oxoiron group embedded in a heme. 2 The peroxidases differ from other heme enzymes such as the cytochrome P450 enzymes in the nature of the axial ligand bound to iron, namely the active species in P450 enzymes is bound to the protein backbone via a thiolate linkage of a cysteinate residue, whereas peroxidases generally bind to a histidine side chain. 3 The differences in axial ligand result in different chemical properties of the active species, i.e., a cysteinate ligand exerts a push effect on the iron, while histidine withdraws electron density. 4 As a result, these two ligands give differences in electronic as well as catalytic properties to the enzyme active site. 5,6 Attempts to mutate the axial ligand of CcP into cysteinate were unsuccessful and resulted in oxidation of the ligand into cysteic acid. 7 In the past we extensively studied the axial ligand effect on the catalytic properties of oxoiron porphyrin models using theoretical methods. 4c,6 In line with this, in this work we present the effect of replacing one or more nitrogen atoms of the histidine axial ligand by phosphorus atoms and the changes this exerts on the catalyst. Recent studies on R-iminophospholide ligands in coordination chemistry showed extensive charge delocalization from the phosphorus atom. 8 Moreover, biphosphines were shown to have strong π-accepting properties and as a result stabilize transition metals in low oxidation states. 9 To find out what effects the replacement of a nitrogen atom by a phosphorus atom has on the electronic and catalytic properties of HRP CpdI, we have investigated models of CpdI with only the axial bound nitrogen atom replaced by phosphorus (CpdI(1P)) and a model in which both nitrogen atoms of the imidazole ring are replaced by phosphorus (CpdI(2P)). The model of the active species of peroxidase CpdI is an oxoiron porphyrin (without side chains) that is bound to an axial imidazole group, and the overall charge of the model is +1. All geometries were fully optimized with Jaguar 5.5 using the UB3LYP hybrid density functional method. 10,11 We employed an LACVP basis set for the geometry optimizations and ran single point calculations with an LACV3P+* basis set to confirm the energetics. 12 All energetics reported in this work are calculated at the LACV3P+* level of theory with ZPE corrections at the LACVP level. The charge distributions were calculated with the Natural Bond Analysis (NBO) program as implemented in Gaussian-03. 13,14 Figure 1 shows the optimized geometries and group spin densities (F) of CpdI(1P) and CpdI(2P) in the lowest lying quartet and doublet spin states. Similarly to CpdI of peroxidase and P450 models, CpdI(1P) and CpdI(2P) are also described as a triradicaloid system: two unpaired electrons located in π* orbitals along the FeO bond and the third one in a nonbonding heme orbital with a 2u symmetry. 5,15 These three electrons are either ferromagnetically coupled into an overall quartet spin state or antiferromagnetically coupled into a doublet spin state. As the coupling between the π* FeO and a 2u electrons is weak, the doublet-quartet energy gap is small: ΔE+ZPE )-0.2 kcal mol -1 in CpdI(1P) and +0.1 in CpdI(2P), where a minus sign implies a doublet spin ground state. These energy differences are virtually identical to those obtained for HRP CpdI, where an energy gap of +0.1 kcal mol -1 was obtained using the same methods and basis sets. The group spin densities match those obtained for HRP CpdI closely, the only minor difference being a slightly higher absolute value on the axial ligand here. 5b In P450 models also a significant axial ligand spin density was obtained due to mixing of the a 2u orbital with a σ S orbital on the ligand. 4c It appears that the phosphorus substituted structures have similar mixing patterns but to a somewhat lesser degree than thiolate ligated systems. The optimized geometries of CpdI(1P) and CpdI(2P) are similar to those obtained for either HRP or P450 CpdI. 5,15 The Fe-O distances are closer to those obtained for P450 CpdI than those for HRP CpdI, where values of 1.651 (1.648) Å and 1.621 * Corresponding author. E-mail: sam.devisser@manchester.ac.uk. 20759 2006, 110, 20759-20761 Published on Web 09/27/2006 10.1021/jp065660q CCC: $33.50 © 2006 American Chemical Society