Cytochrome P450 Reactivity Is Finally Explained

The active sites of Cyt. P450s contain a single heme b cofactor with a proximal cysteinate ligand. Figure 1 shows the active site of P. putida Cyt. P450cam as an example. The catalytic mechanism of Cyt. P450s has been studied in detail and is depicted in Figure 1. After one-electron reduction of the heme, O2 is bound first and subsequently reduced and protonated, generating an Fe–OOH complex (Compound 0). Protonation of this species results in heterolytic O O bond cleavage, producing the critical intermediate Compound I and H2O. Compound I is able to abstract a hydrogen atom from the substrate resulting in an Fe– OH complex (Compound II) and a carbon-based substrate radical, according to the “rebound” mechanism. The carbon-based radical is finally hydroxylated to complete the catalytic cycle. Although Cyt. P450 enzymes have been studied for the past 56 years, key questions have remained with respect to the nature of Compound I, its ability to oxidize substrates, and the role of the proximal thiolate ligand for catalysis. Progress in this regard has been stalled by the difficulty in trapping Compound I, which is usually short-lived and generated only in low yield. Recently, however, Green and co-workers obtained Compound I in high yield ( 75%) for the first time by reacting a thermophilic Cyt. P450 (CYP119) in its ferric oxidation state with meta-chloroperoxybenzoic acid (mCPBA). The UV/Vis spectrum of this species, which is stable in solution for roughly 35 ms, exhibits the Soret band at 367 nm and Q bands at 610 and 690 nm (Figure 2). Rapidfreeze quench Mçssbauer spectroscopy of this intermediate shows isomer shifts and quadrupole splitting values (d= 0.11 mms 1 and DEQ= 0.90 mms ) similar to those of Compound I of chloroperoxidase (CPO), confirming the Fe oxidation state of iron. The EPR spectrum of Compound I exhibits g? values of 1.86 and 1.96 and gk= 2.0, which is indicative of an S= =2 system. This suggests that the Fe =O unit (S= 1) in this species is antiferromagnetically coupled to the porphyrin-based radical (S= =2), leading to a doublet (S= =2) ground state for Compound I. [3a] These findings are in agreement with the results of DFT and QM/MM calculations. Figure 1. Top: O2 bound to the ferrous heme in the active site of Cyt. P450cam from P. putida (PDB code 1DZ9). Bottom: The catalytic cycle of Cyt. P450 monooxygenases (where R–H is the substrate).