ET from Trp171 to Cpd I in the absence of tartaric acid. To decipher the mechanism of the first ET step (ET from Trp171 to Cpd I), multiscale simulations have been carried out. First, our classical MD simulations indicated that the sidechain N atom of Trp171 keeps a distance of ~16.6 Å from the Fe atom in the heme group (see Figure ), which distance is reasonable for biological electron transfer. Then, we calculated the ET pathway which connects the Trp171-N atom and Heme-Fe atom based on a representative structure by using the Pathways plugin in the VMD software. As shown in Figure , the most favorable ET pathway between these two atoms is Trp171→Met172→Leu173→His176→ Heme. Based on the same representative structure from the classical MD simulations we carried out the QM/MM MD simulations, in which we selected the QM regions based on the ET pathway analysis mentioned above. During the QM/MM MD simulations, we can track the electron transfer process according to the calculated spin densities. Interestingly, QM/MM MD simulation shows that an electron can transfer from Trp171 to Cpd I within ~300 fs, suggesting that the first ET step happens spontaneously. This is consistent with our QM model calculations, showing that the first ET step, which generates a Trp171 cation radical, is exoergic by 1.77 kcal/mol (see Figure ). Moreover, further QM/MM MD simulation leads to a fast PT from the Trp171 cation radical to Glu250 within ~620 fs, yielding a Trp171 neutral radical and protonated Glu250 (see Figure ). This result suggests that the long-range ET from Trp171 to Cpd I follows a stepwise ETPT mechanism, in which the ET is followed by PT 2, 3 . However, as noted in our previous study, the QM/MM MD simulations may significantly overestimate the rate of an ET reaction, mostly due to the neglection of the electronic transmission coefficient in the Marcus theory. Thus, the ET rate constant was later estimated by using the Marcus theory in the present study. ET from the lignin substrate to Trp171 in the absence of the tartaric acid. In the last section, our QM/MM MD simulations suggest the ET from Trp171 to Cpd I follows a stepwise ETPT mechanism, yielding a Trp171 neutral radical and Cpd II. Then, Cpd II would undergo a fast protonation to form the Fe(IV)-OH species, while the Trp171 neutral radical would oxidize the lignin substrate. Unlike the first ET step, we did not observe a spontaneous ET from the lignin substrate to Trp171 during the QM/MM MD simulation, suggesting the second ET step may be thermodynamically unfavorable. This agrees with our QM model calculations which showed that the second ET is endoergic by 4.06 kcal/mol (See Figure ). As the protonated Glu250 forms a persistent H-bond with Trp171 during QM/MM simulations, we assume it may back donate the proton to the Trp171 neutral radical, which may drive the long-range ET from the lignin substrate to the Trp radical via a PCET mechanism. To verify such possibility, QM/MM metadynamics simulations were carried out to study the thermodynamics of this PCET process, with the calculated free energy profile depicted in Figure . It indicated that the PCET happens through a PT from Glu250 to Trp171, which is coupled with the ET from the lignin substrate to Trp171, yielding an in-tact Trp171 and a lignin cation radical. This process involves a free energy barrier of ~7.5 kcal/mol and a free energy change of ~2 kcal/mol. Once the lignin cation radical was generated, it may undergo C-C cleavage spontaneously to afford the lignin fragments. Our calculations showed that the C-C cleavage has a free energy barrier of 10.76 kcal/mol and is endoergic by 5.47 kcal/mol. (Figure ) Thus, the overall reaction for C-C cleavage is endoergic.