Introduction 1 2 3 4 5 1 6 7 8 9 9 10 11 12 12 17 9 14 17 Fig. 1 a C443 b c green blue red white orange  18 19 19 20 21 19 22 27 11 28 29 30 31 31 35 Materials and methods Materials 6 6 Escherichia coli Expression and purification of the enzymes E. coli 6 6 36 RR spectroscopy K s 6 37 −1 Computational details Model 5 38 − Results Discussion Method 39 41 s 2 s 2 s 2 p 6 42 43 32 5 water Thr α meso The energy profiles for the mutant enzyme model were obtained by the same procedure for each structure of the wild-type profile in which the threonine hydroxyl moiety was replaced by a methyl substituent. Results Enzymes expression and purification, UV–vis absorption spectroscopy E. coli 2 Fig. 2 solid line dashed line  RR spectroscopy 3 −1 ν 4 19 Fig. 3 WT T309V Methods  4 27 −1 ν 3 ν 2 ν 10 19 ν 3 −1 ν 3 ν 2 ν 10 Fig. 4 WT T309V Methods solid lines dotted lines bold  4 ν 3 ν 2 ν 10 ν 3 ν 2 −1 ν 3 ν 2 −1 5 ν 3 −1 Fig. 5 WT T309V solid lines dotted lines ν 3  ν Fe–CO −1 6 27 ν Fe–CO −1 ν Fe–CO Fig. 6 WT T309V DX  DFT calculations 1 1 −1 1 −1 7 −1 water Thr position 2 1 Fig. 7 filled circles filled squares dashed line open circles open squares LS HS 5c 6c  7 −1 water Thr 1 π Table 1 E π  Fe–CO Fe–C≡O Wild type Mutant Wild type Mutant Distance (Å) 1.824 1.851 1.147 1.146 E −1 −24.6 −24.2 – – Mayer bond order 0.21 0.20 0.52 0.52 π – – e − e − The superimposed geometries of the fully optimized and experimental structures, of the partially optimized structures of the T309V mutant with water in positions 1 and 2, and of the fully optimized structures of the CO-bound species for both the wild type and the T309V mutant are available as supplementary material. Discussion 3+ trans 44 5 1 1 45 47 dπ π 48 49 9 14 1 4 50 53 5 44 ν 3 5 6 54 12 14 17 55 56 12 14 In an effort to rationalize the experimental observations, we looked for an explanatory model, compatible with our spectroscopic data, which could be tested using available quantum-mechanical methods. The absence of direct interactions between T309 and the axial ligand indicates that T309 exerts its influence on the spin equilibria of CYP2D6 indirectly. 13 14 1 7 Although the computational model of the CYP2D6 active site used in the present study only includes a few amino acids and the iron–porphyrin complex, and therefore does not take into account the rest of the protein, it provides a clear rationale for the observed experimental data. Unfortunately, an evident limit of this model is the inability to include the substrate and to study its influence on the spin equilibrium of the substrate-bound enzymes, for which extended models and methods have to be used. However, it should be stressed that experimental data indicate that the role of T309 in the CYP2D6 spin equilibrium is independent of that of DX; therefore, it is reasonable to assume that the implications of our model for the role of T309 would still be valid for the substrate-bound enzymes. Conclusions On the basis of RR data, it is concluded that the T309V mutant of CYP2D6 has an altered spin equilibrium with respect to the wild-type enzyme, with a relative higher amount of 6cLS species at the expense of the 5cHS species, in both the resting state and the substrate-bound forms. Apparently, there is no direct interaction between residue T309 and the heme sixth ligand, suggesting an indirect mechanism of action on the spin equilibrium. Spectroscopic data also indicate that the T309V mutation does not significantly alter the polarity of the heme environment, excluding an increased number of water molecules in the heme pocket as the reason for the altered spin state. DFT calculations show that a simple model, involving a water molecule alternatively occupying two positions inside the heme pocket corresponding to two different spin states, is able to explain the experimental data. In this model, the position corresponding to the 5cHS state is stabilized by a hydrogen bond with T309, and a T309V mutation will induce an increase of the 6cLS species, as experimentally observed. Electronic supplementary material Below is the link to the electronic supplementary material.  Supplementary material (PDF 215 kb)