1. Volume 3, Issue 1, Pages 41-62 (January 1995)
Structure and function of cytochromes P450:a comparative analysis of three crystal structures 
Charles A Hasemann, Ravi G Kurumbail, Sekhar S Boddupalli, Julian A Peterson, Johann Deisenhofer 
Structure 
Volume 3, Issue 1, Pages (January 1995)
DOI: /S (01)

2. Figure 1
Similarity of the overall fold and secondary structure content of the three P450s. The molecules are viewed from the substrate access (distal) face. α–Helices are presented as purple coils, 310–helices and π–helices as green coils, β –strands as red arrows, and loops as yellow tubes. The heme molecules are shown as ball-and-stick models. The visible secondary structures of each molecule are labeled (α–helices A, B′ , D–G, I and L, and β–sheets β 1–β 5), as are the amino (N) and carboxyl (C) termini. (Figure prepared using the program RIBBONS [91] ) .
Structure 1995 3, 41-62DOI: ( /S (01) )

3. Figure 2
Plot of the relationship between structural similarity and sequence identity. The test-set data (○) were tabulated by Chothia and Lesk [28] in a study which concluded that the rms difference in the core regions of homologous molecules was predictably correlated to their sequence identity. This relationship is shown to hold true for the P450s in this study (▴) .
Structure 1995 3, 41-62DOI: ( /S (01) )

4. Figure 3
Sequence alignment of P450cam , P450terp and P450BM-3 based on their structural superposition. Gaps in the alignments are indicated by dots, and the position number of the amino acid at the far right of each line is shown. Names of the α–helices and β–strands are indicated above the sequences. The extent of the secondary-structure elements of each sequence are boxed and color-coded (α –helices magenta, 310–helices green, π–helices orange, β–strands red, and the Cys-pocket in blue). Note that this amino acid alignment is based on the actual spatial superposition of the structures, and not optimization of sequence conservation .
Structure 1995 3, 41-62DOI: ( /S (01) )

5. Figure 4
Cα trace of P450BM-3 . Stereo pairs of the model are shown, as viewed from both the distal (a) and proximal (b) sides. The tube is colored according to the rms deviation of the Cα at each position in the three structures as aligned in Figure 3 . The transition from cool to warm colors (dark blue to light blue to green to yellow to orange and red) represents the progression of structural conservation from most similar to most variable. The backbone of P450BM-3 was chosen for this figure because it contains the largest number of insertions relative to either P450cam or P450terp . Regions of P450BM –3 which are insertions were assigned the highest score, and thus appear red. The amino (N) and carboxyl (C) termini are labeled .
Structure 1995 3, 41-62DOI: ( /S (01) )

6. Figure 4
Cα trace of P450BM-3 . Stereo pairs of the model are shown, as viewed from both the distal (a) and proximal (b) sides. The tube is colored according to the rms deviation of the Cα at each position in the three structures as aligned in Figure 3 . The transition from cool to warm colors (dark blue to light blue to green to yellow to orange and red) represents the progression of structural conservation from most similar to most variable. The backbone of P450BM-3 was chosen for this figure because it contains the largest number of insertions relative to either P450cam or P450terp . Regions of P450BM –3 which are insertions were assigned the highest score, and thus appear red. The amino (N) and carboxyl (C) termini are labeled .
Structure 1995 3, 41-62DOI: ( /S (01) )

7. Figure 5
Multiple sequence alignment of various P450s based primarily on sequence conservation. Using the correctly aligned sequences of P450terp , P450cam and P450BM-3 , other P450s were aligned in order to maximize sequence alignment, without violating conserved motifs observed in the crystal structures. The approximate locations of the secondary structures, the Cys-pocket and the meander regions in the three known crystal structures are shown above the sequences. Groups of 10 amino acids are separated by spaces, and the alignment position at the end of each row is shown. The single letter amino acid code is used, with gaps in individual sequences indicated by a dot. The consensus sequence is presented as follows: upper-case letter, 100% conservation; lower-case letter, most frequently occurring amino acid in ≥ 25% of the sequences; dot, no individual amino acid in ≥ 25% of the sequences, or two or more amino acids at an equal frequency in ≥ 25% of the sequences. The trivial name, standard nomenclature, and source organism for the sequences presented are: P450cam (CYP101, P. putida ), P450terp (CYP108, Pseudomonas sp. ), 11A1_Bovin (P450scc , CYP11A1, bovine), 27_Rabbit (P45026-ohp , CYP27, rabbit), 21a2_human (P450c21B , CYP21A2, human), 17a_Rat (P45017α , CYP17, rat), 1a2_Rat (P450d , CYP1A2, rat), 2c11_Rat (P450M1 , CYP2C11, rat), 2c4_Rabbit (P450PBc4 , CYP2C4, rabbit), 2e1_rabbit (P4503a , CYP2E1, rabbit), 2b1_Rat (P450b , CYP2B1, rat), 2a4_Mouse (P45015α oh-1 , CYP2A4, murine), 3a4_human (P450nf-25 , CYP3A4, human), 4a1_rat (P450LAω , CYP4A1, rat), P450BM-3 (CYP102, B. megaterium ), 19_Human (P450Arom , CYP19, human). The source of the amino acid sequences, and the method of alignment are provided in the Materials and methods section.
Structure 1995 3, 41-62DOI: ( /S (01) )

8. Figure 6
Stereoview of the conserved amino acids involved in heme binding, shown for P450terp . The β–bulge known as the Cys-pocket is shown as a pink tube. The invariant cysteine ligand to the heme iron, and the invariant phenylalanine which completes the hydrophobic shielding of the cysteine–iron bond, are shown. Amino acids that are involved in propionate coordination are also shown. Two of these are found in the C helix (pink coil): His110 is a conserved hydrogen-bond donor (position 175, Figure 5 ), and Arg114 (position 179, Figure 5 ) is a conserved basic residue which forms a salt bridge to the D-ring propionate. The Cys-pocket amino acid at position 375 (His375, position 524, Figure 5 ), and the main-chain nitrogen at position 372 (position 521, Figure 5 ) interact with the D-ring and A-ring propionates in all three structures. The conserved arginine (Arg319, position 453, Figure 5 ) in β–strand 1-4 (pink arrow) forms a salt bridge with the A-ring propionate, and a water (red sphere) hydrogen bonds with that propionate and the amino acid at position 72 of P450terp (Asn72, position 135, Figure 5 ) .
Structure 1995 3, 41-62DOI: ( /S (01) )

9. Figure 7
Stereoviews of the meander region and the conserved ERR-triad. (a) Cα trace showing the superposition of the meander regions of P450terp (green), P450cam (red) and P450BM-3 (purple). For the purpose of this Figure, the structures were superimposed by optimizing the fit of Cα s in the region between the K′ helix and the Cys-pocket (positions 482–518, Figure 5 ) as well as the Cα s of the conserved glutamate and arginine side chains in the K helix (positions 439 and 442, Figure 5 ). Side-chain atoms of the conserved glutamate and arginine in the K helix, and the conserved arginine in the meander (position 498, Figure 5 ) are also shown. (b) Detail of the interactions that stabilize the recurring structure of the meander regions, shown for P450terp . The Cα trace of the meander (magenta tube) and a portion of the K helix (magenta coil) are shown. Salt-bridge interactions between the glutamate (Glu306) and arginine (Arg309 and Arg362) side chains, and hydrogen bonds between the arginine side chains and meander carbonyl oxygens are indicated by narrow lines. The Cα s of positions 353 and 357 are numbered.
Structure 1995 3, 41-62DOI: ( /S (01) )

10. Figure 7
Stereoviews of the meander region and the conserved ERR-triad. (a) Cα trace showing the superposition of the meander regions of P450terp (green), P450cam (red) and P450BM-3 (purple). For the purpose of this Figure, the structures were superimposed by optimizing the fit of Cα s in the region between the K′ helix and the Cys-pocket (positions 482–518, Figure 5 ) as well as the Cα s of the conserved glutamate and arginine side chains in the K helix (positions 439 and 442, Figure 5 ). Side-chain atoms of the conserved glutamate and arginine in the K helix, and the conserved arginine in the meander (position 498, Figure 5 ) are also shown. (b) Detail of the interactions that stabilize the recurring structure of the meander regions, shown for P450terp . The Cα trace of the meander (magenta tube) and a portion of the K helix (magenta coil) are shown. Salt-bridge interactions between the glutamate (Glu306) and arginine (Arg309 and Arg362) side chains, and hydrogen bonds between the arginine side chains and meander carbonyl oxygens are indicated by narrow lines. The Cα s of positions 353 and 357 are numbered.
Structure 1995 3, 41-62DOI: ( /S (01) )

11. Figure 8
Substrate ‘footprints’ for the three P450s. The Cα trace of each molecule is shown in blue, with the atoms of substrate-contact residues highlighted. The heme molecules are also shown (in pink). The identity of contact residues is based on the camphor-bound form of P450cam [13] , on docking studies [22] and preliminary analysis of an α–terpineol-bound form of P450terp (CA Hasemann and J Deisenhofer, unpublished data), and on preliminary analysis of arachidonate [23] and laurate (SS Boddupalli, RG Kurumbail and J Deisenhofer, unpublished data) bound forms of P450BM-3.
Structure 1995 3, 41-62DOI: ( /S (01) )

12. Figure 9
Comparison of the variable regions of the three P450s which might be involved in substrate recruitment and binding. P450terp (green), P450cam (red), and P450BM-3 (magenta) were superimposed as described for Table 3 and Figure 4 . A stereo pair of the Cα trace is shown for each molecule as superimposed. The heme of P450terp is shown in order to demonstrate the proximity of these structures to the site of catalysis. (a) B′ helices and flanking polypeptide segments. The size and spatial disposition of each helix is quite different, as are the lengths of the polypeptide segments that surround them. (b) F and G helices, and the F-G loop. The orientation of this view is the same as for part (a). The G helices of P450terp and P450BM-3 clearly follow a different trajectory from that of the G helix of P450cam . This leaves them further from the heme, out of the region which might play a direct role in substrate binding. Similarly, the shorter F helix and F-G loop of P450cam is much closer to the heme than observed for P450BM-3 , forming a tighter ‘lid’ over the substrate-access channel. Note that the loop between the F and G helices of P450terp was disordered in the crystal, and thus was not modeled .
Structure 1995 3, 41-62DOI: ( /S (01) )

13. Figure 9
Comparison of the variable regions of the three P450s which might be involved in substrate recruitment and binding. P450terp (green), P450cam (red), and P450BM-3 (magenta) were superimposed as described for Table 3 and Figure 4 . A stereo pair of the Cα trace is shown for each molecule as superimposed. The heme of P450terp is shown in order to demonstrate the proximity of these structures to the site of catalysis. (a) B′ helices and flanking polypeptide segments. The size and spatial disposition of each helix is quite different, as are the lengths of the polypeptide segments that surround them. (b) F and G helices, and the F-G loop. The orientation of this view is the same as for part (a). The G helices of P450terp and P450BM-3 clearly follow a different trajectory from that of the G helix of P450cam . This leaves them further from the heme, out of the region which might play a direct role in substrate binding. Similarly, the shorter F helix and F-G loop of P450cam is much closer to the heme than observed for P450BM-3 , forming a tighter ‘lid’ over the substrate-access channel. Note that the loop between the F and G helices of P450terp was disordered in the crystal, and thus was not modeled .
Structure 1995 3, 41-62DOI: ( /S (01) )

14. Figure 10
The central region of the I helices of P450terp and P450BM-3 are more similar to one another than to that of P450cam . Cα traces of the central I helices of P450terp (green), P450cam (red) and P450BM-3 (magenta) are shown in stereo with the heme of P450terp . While all three helices have a disruption in the normal helical hydrogen-bonding pattern which results in a slightly ‘untwisted’ helix in this region, the location of this distortion is different in P450cam compared with P450terp and P450BM-3 . For this figure the three molecules were superimposed to minimize the deviation between Cα positions in the central I helices only (positions 373–393, Figure 5 ). This was done to assure that no differences in distant regions of the molecule would affect the superposition of this region.
Structure 1995 3, 41-62DOI: ( /S (01) )

15. Figure 11
Features of the central I helices of P450cam and P450terp which may be involved in the delivery of catalytic protons for oxygen scission. Stereo pairs of the I and F helices are shown as magenta coils, with important side-chain and main-chain atoms included. The hemes are presented as ball-and-stick models, with the heme iron represented by a green sphere, and the axial ligand to the heme (either water or hydroxide) shown as a red sphere. Several other relevant waters are also represented as red spheres. Hydrogen-bond and salt-bridge interactions are indicated with solid white lines. (a) Atomic coordinates for the substrate-free form of P450cam were used for this figure [12] , so that the disposition of the distal axial ligand (hydroxide or water) could be shown. The disruption of normal helical hydrogen bonds is at the ‘back’ of the helix as viewed here. The hydrogen bond between the invariant threonine (Thr252) and the carbonyl oxygen of Gly248 is shown, as is the hydrogen bond from the carbonyl oxygen of Gly249 to the water which has intercalated into the ‘back’ of the helix. The conserved acid side chain (Asp251) is also shown, forming salt bridges to Arg186 and Lys178, and hydrogen bonding to Thr185. The water molecules adjacent to Arg186 and Lys178 are at the exterior of the molecule, and represent the closest approach for bulk solvent-derived protons to the central I helix. (b) The I helix of P450terp , viewed from the same perspective as for P450cam in (a). In contrast to P450cam , the disruption of helical hydrogen bonds is at the ‘front’ of the helix. The hydrogen bond between the invariant threonine (Thr271) and the carbonyl oxygen of Ala267 is shown, as are the hydrogen bonds from the carbonyl oxygen of Thr266 and the main-chain nitrogen of Asp270 to the waters which have intercalated into the ‘front’ of the helix. Note also that there is a hydrogen bond between the carbonyl oxygen of Ala267 and the axial ligand (water or hydroxide) to the heme iron. The conserved acidic side chain (Asp270) is also shown, forming a salt bridge with Lys419, and hydrogen bonding to Gln185. As shown for P450cam , the water molecules adjacent to Lys419 are at the exterior of the molecule, and again represent the nearest source of bulk solvent protons for catalysis.
Structure 1995 3, 41-62DOI: ( /S (01) )

16. Figure 11
Features of the central I helices of P450cam and P450terp which may be involved in the delivery of catalytic protons for oxygen scission. Stereo pairs of the I and F helices are shown as magenta coils, with important side-chain and main-chain atoms included. The hemes are presented as ball-and-stick models, with the heme iron represented by a green sphere, and the axial ligand to the heme (either water or hydroxide) shown as a red sphere. Several other relevant waters are also represented as red spheres. Hydrogen-bond and salt-bridge interactions are indicated with solid white lines. (a) Atomic coordinates for the substrate-free form of P450cam were used for this figure [12] , so that the disposition of the distal axial ligand (hydroxide or water) could be shown. The disruption of normal helical hydrogen bonds is at the ‘back’ of the helix as viewed here. The hydrogen bond between the invariant threonine (Thr252) and the carbonyl oxygen of Gly248 is shown, as is the hydrogen bond from the carbonyl oxygen of Gly249 to the water which has intercalated into the ‘back’ of the helix. The conserved acid side chain (Asp251) is also shown, forming salt bridges to Arg186 and Lys178, and hydrogen bonding to Thr185. The water molecules adjacent to Arg186 and Lys178 are at the exterior of the molecule, and represent the closest approach for bulk solvent-derived protons to the central I helix. (b) The I helix of P450terp , viewed from the same perspective as for P450cam in (a). In contrast to P450cam , the disruption of helical hydrogen bonds is at the ‘front’ of the helix. The hydrogen bond between the invariant threonine (Thr271) and the carbonyl oxygen of Ala267 is shown, as are the hydrogen bonds from the carbonyl oxygen of Thr266 and the main-chain nitrogen of Asp270 to the waters which have intercalated into the ‘front’ of the helix. Note also that there is a hydrogen bond between the carbonyl oxygen of Ala267 and the axial ligand (water or hydroxide) to the heme iron. The conserved acidic side chain (Asp270) is also shown, forming a salt bridge with Lys419, and hydrogen bonding to Gln185. As shown for P450cam , the water molecules adjacent to Lys419 are at the exterior of the molecule, and again represent the nearest source of bulk solvent protons for catalysis.
Structure 1995 3, 41-62DOI: ( /S (01) )

17. Figure 12
Electrostatic potential at the molecular surfaces of (a) P450terp , (b) P450cam and (c) P450BM-3 . The consistent positively charged (blue) patch near the center of the face of each molecule is located directly above the heme, and is a good candidate for the docking site between a P450 and its electron-donating redox partner. Molecular surfaces were color-coded by an interpolated value of the electrostatic potential at a point as calculated in the program GRASP [81] . Surface points are colored in a spectrum from blue to red corresponding to positive and negative potential respectively, with neutral points colored white. The deepest shades of blue and red correspond to potentials of ≥ +6.0 kcal and ≥–6.0 kcal respectively .
Structure 1995 3, 41-62DOI: ( /S (01) )

18. Figure 12
Electrostatic potential at the molecular surfaces of (a) P450terp , (b) P450cam and (c) P450BM-3 . The consistent positively charged (blue) patch near the center of the face of each molecule is located directly above the heme, and is a good candidate for the docking site between a P450 and its electron-donating redox partner. Molecular surfaces were color-coded by an interpolated value of the electrostatic potential at a point as calculated in the program GRASP [81] . Surface points are colored in a spectrum from blue to red corresponding to positive and negative potential respectively, with neutral points colored white. The deepest shades of blue and red correspond to potentials of ≥ +6.0 kcal and ≥–6.0 kcal respectively .
Structure 1995 3, 41-62DOI: ( /S (01) )

19. Figure 12
Electrostatic potential at the molecular surfaces of (a) P450terp , (b) P450cam and (c) P450BM-3 . The consistent positively charged (blue) patch near the center of the face of each molecule is located directly above the heme, and is a good candidate for the docking site between a P450 and its electron-donating redox partner. Molecular surfaces were color-coded by an interpolated value of the electrostatic potential at a point as calculated in the program GRASP [81] . Surface points are colored in a spectrum from blue to red corresponding to positive and negative potential respectively, with neutral points colored white. The deepest shades of blue and red correspond to potentials of ≥ +6.0 kcal and ≥–6.0 kcal respectively .
Structure 1995 3, 41-62DOI: ( /S (01) )

20. Figure 13
Cartoon representation of the ‘molecular dipole’ for P450BM-3 . A Cα trace is shown, with a plane cutting through the mid-region of the molecule. The plane is colored by an interpolated value for the electrostatic field on a grid at the surface of the plane. The charge separation that leads to the molecular dipole (yellow arrow) is evident. The proximal (redox-partner docking) and distal (substrate access) faces of the protein are indicated .
Structure 1995 3, 41-62DOI: ( /S (01) )