Fig. 1. All calculated channels mapped on to the structure of cytochrome P450cam (PDBid: 1AKD). The protein is shown in a shaded gray cartoon representation. The mouths of the channels are shown as circles located at the corresponding position on the protein surface. The channels that share the same color are related by: (i) their opening modes (2c, 2ac, 2a, 2f, 2b—red; 2e, 2d, 4—green; 1, 3—brown), (ii) function [solvent (S), water (W)—blue], or (iii) being newly identified (5—violet). Each channel is shown in a different nuance of the shared color. The B–C loop is colored pink while the F–G loop is green. Alpha-helices are annotated with their corresponding letters. The heme cofactor is shown in a “Licorice” representation colored by atom type. Two distinct views (related by a ca. 90° rotation towards the viewer and a ca. 180° rotation in the paper plane) were required to show all identified channels: (A) side-view looking down the I helix; (B) top-view looking down on the heme cofactor from the distal side.

Fig. 2. Examples of different cytochrome P450 topologies with the identified channels. The representations and views are the same as in Fig. 1, except that the channels are shown using the CAVER-generated van der Waals representation in red. (A) Bacterial CYP152A2 (PDBid 2D09); (B) bacterial P450-BM3 (PDBid 1JPZ); (C) mammalian CYP2C8 (PDBid 1PQ2); (D) bacterial CYP152A1 (PDBid 1IZO); (E) archaeal CYP119 (PDBid 1F4T); (F) mammalian CYP2C5 (PDBid 1NR6).

Fig. 3. Curvy 2e channel in the bacterial CYP107A1 (PDBid 1JIN). (A) CAVER representation of the 2e channel (view analogous to those in Fig. 2). The view through the channel to the active site not easily visible from the molecular surface.

Fig. 4. The relative position of the FG and BC loops determines the degree of opening of channel 2a and the number and nature of channels that merge in an open structure. The protein is shown in a wireframe MSMS molecular surface representation, with the F–G and B–C loops highlighted as a solid surface colored in green and pink respectively. The view is a top view of channel 2a (see Fig 1). (A) Discrete opening of channel 2a in bacterial CYP152A1 (PDBid 1IZO); (B) crevice formed by the merging of channels 2c, 2ac, 2a, 2f and S in the fungal P450 NOR (PDBid 1CL6); (C) large crevice or funnel formed by the merging of channels 2c, 2ac and 2a in the mammalian CYP2B4 (PDBid 1PO5).

Fig. 5. Different profiles of the solvent (S) channel. The representations are the same as in Fig. 2, except that a close-up of the region of the active site is shown. (A) Narrow S channel, permitting the passage of a water probe in the bacterial P450-BM3 (PDBid 2BMH); (B) S channel with 2 branches, each of them permitting the passage of a water probe, in the mammalian CYP2C5 (PDBid 1NR6); (C) wide S channel in the mammalian CYP2D6 (PDBid 2F9Q) that might permit the passage of substrates or products.

Table 1.

Occurrence of open active site access channels in all crystal structures of cytochrome P450s available in March 2006

^a The number of PDB files and protein chains for which results were obtained.
^b Structures were classified as closed (C), discrete (D) or merged (M), indicating crystal structures with, respectively, no solvent accessible channels, defined discrete channels, or two or more channels merged to form one channel. In the latter case, there may be further discrete channels in addition to the merged channel.
^c The channels are as shown in Fig. 1. and defined in Section 2. ‘S’ indicates the solvent channel and ‘W’ the water channel. The number in each column is the number of chains observed to contain the named channel. 2c, 2ac, 2a, 2f and the solvent channel are arranged in the order listed in an arc around the F–G helix–loop–helix, and have the possibility of merging; 2b may also merge with 2a. Channels which are observed to merge are marked as indicated in the following examples: ▪4 four channels, one or more merge with the channel to the left (excepting 2b which merges with 2a), but not the one to the right. 3▪ three channels, one or more merge with the channel to the right, but not to the left. ▪3▪ three channels, one or two merge with a channel to the left but not the right, one or two merge with a channel to the right but not the left. ▪4▪ four channels, one or more merge with the channels to the right and left.
^d 1IO7A is missing the F–G loop and appears to have merged 2a and 2f for this reason. 1IO7B is complete and open with merged channels.
^e PDB entry 1UE8. A P450 from Sulfolobus tokodaii. A Blast sequence alignment indicates 63% sequence identity to CYP119 from Sulfolobus solfactaricus.
^f 2BDM has 2 discrete 2e channels, one near the B' helix and one near the plane of the heme.
^g This structure is missing the F–G loop and for this reason appears wide open.
^h 1LGF has many missing structural elements and is responsible for the large merged channel indicated, although some merging is seen in the other channels.
ⁱ Both chains are missing the F–G and B–C loops; the observed channels are a result of this.
^j PDB entry 1WIY. This structure is from Thermus thermophilusand a Blast sequence alignment indicates 94% sequence identity with CYP175A1 from the same organism.