P450cam and its Reductase System

The most intensively studied class I P450 redox system is P450cam along with its PdR and Pd redox partners. All components of the system are soluble proteins and interact with one another in the cytosol of Pseudomonas putida. The atomic structures of all three components of the system have been determined, and the P450 component has been structurally characterized in a number of different substrate- and ligand-bound forms [e.g. 9, 10, 20, 21]. PdR is a 48 kDa FAD-binding protein which is reduced by NADH. Pd is a 11 kDa 2Fe-2S cluster-containing fer-redoxin (Fig. 5.1b). The system couples NADH oxidation tightly to the formation of 5-exo-hydroxycamphor, with Pd acting as a single electron carrier between the reductase and the P450. Substrate (camphor) binding to P450cam results in modulation of the heme iron spin-state equilibrium, with the P450 converting from a predominantly low-spin ferric form to a predominantly high-spin form at substrate saturation [22]. A large positive shift in heme iron reduction potential (~130 mV from ca. -300 mV to -170 mV) accompanies this conversion, acting as a thermodynamic trigger for electron transfer from Pd to the substrate-bound P450 [23].

The steady-state turnover of P450cam in camphor hydroxylation is rate-limited by electron transfer from (but not binding of) Pd. At high Pd concentrations the first electron transfer reaction to ferric P450cam is rate-limiting (measured at ~15 s-1 at 4 °C), but at more physiologically relevant Pd concentrations the second electron transfer to the P450 becomes rate-limiting [8, 24]. Chemical modification studies of Pd indicated that a cluster of acidic residues is involved in interactions critical for electron transfer from PdR. Thus electrostatic binding is considered as important for productive PdR/Pd interactions [25]. The acidic cluster is in the same protein region as that recognized to be important for binding of the mammalian adrenal gland mitochondrial 2Fe-2S ferredoxin adrenodoxin to its reduc-tase partner adrenodoxin reductase [26]. Cytochrome b5, a known effector (and likely second electron donor) for certain mammalian steroidogenic P450s, was found to bind tightly to P450cam, with binding mediated by electrostatic interactions between basic residues on the proximal heme face of the P450 and acidic residues on the b5 [27-29].

Pd competitively inhibits binding of b5 to P450cam, and this is consistent with a binding site for the redox partner close to the heme of the P450, and compatible with efficient electron transfer between the proteins [27, 30]. However, it is not clear to what extent the b5/Pd sites on P450cam overlap. What is clear is that a tryptophan residue at the C-terminus of Pd (Trp 106) is needed for efficient electron transfer to P450cam. However, removal of this residue leads to diminished binding (higher Kd value) of Pd to P450cam, and there is no proof that Trp 106 participates directly in the electron transfer process [31-33].

Cytochrome P450cam (formally CYP101A1) was the first P450 enzyme to have its atomic structure resolved, and this structure has provided an important template on which several other P450 structures have been modeled over the years. P450cam has also been structurally resolved in several different substrate/ligand complexes and also, for example, in its reduced/CO-bound complex [e.g. 34]. Only in recent years have the atomic structures of Pd and PdR been resolved from protein crystals [9, 10].

The structure of PdR reveals a fold similar to those of disulfide reductases, and that of Pd is consistent with a key role for the C-terminal residue Trp106 in regulation of electron transfer to P450cam (and possibly from PdR). Structural studies of reduced Pd indicate that conformational changes occur in the protein that likely underlie the marked alterations in affinity previously observed for binding of the oxidized and reduced forms of Pd to P450cam [35]. Modeling of the interactions between PdR and Pd indicate that there is steric complementarity between the two proteins and hint at the possibility of multiple electron transfer pathways between the redox partners [36].

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