Although CYP101D1 and P450cam catalyze the same reaction at similar rates and share strikingly similar active site architectures, there are significant functional differences. CYP101D1 thus provides an opportunity to probe what structural and functional features must be shared and what features can differ but maintain the high catalytic efficiency. Crystal structures of the cyanide complex of wild-type CYP101D1 and it active site mutants, D259N and T260A, have been determined. The conformational changes in CYP101D1 upon cyanide binding are very similar to those of P450cam, indicating a similar mechanism for proton delivery during oxygen activation using solvent-assisted proton transfer. The D259N-CN- complex shows a perturbed solvent structure compared to that of the wild type, which is similar to what was observed in the oxy complex of the corresonding D251N mutant in P450cam. As in P450cam, the T260A mutant is highly uncoupled while the D259N mutant gives barely detectable activity. Despite these similarities, CYP101D1 is able to use the P450cam redox partners while P450cam cannot use the CYP101D1 redox partners. Thus, the strict requirement of P450cam for its own redox partner is relaxed in CYP101D1. Differences in the local environment of the essential Asp (Asp259 in CYP101D1) provide a strucutral basis for understanding these functional differences.

We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDX_D38 which forms an important ion pair with P450cam_R112 at the center of the interface is Arx_L39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with Arx_L39.

A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo-hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme, about 15-20 Å away, Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing are substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV-vis spectroscopy. Individually, the mutants have little effect on activity or structure, but in combination there is a major drop in enzyme activity. This loss in activity is due to the mutants being locked in the low-spin state, which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well-separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states.

Cytochrome P450s are among natures most powerful catalysts. Their ability to activate molecular dioxygen to form high-valent ferryl intermediates (Compounds I and II) enables a wide array of chemistries ranging from simple epoxidations to more complicated C-H bond oxidations. Oxygen activation is achieved by reduction of the ferrous dioxygen complex, which requires the transfer of an electron from a redox partner and subsequent double protonation to yield a water molecule and a ferryl porphyrin π-cation radical (Compound I). Previous studies of the CYP101 family of cytochrome P450s demonstrated the importance of the conserved active site Asp25X residue in this protonation event, although its precise role is yet to be unraveled. To further explore the origin of protons in oxygen activation, we analyzed the effects of an Asp to Glu mutation at the 25X position in P450cam and in CYP101D1. This mutation inactivates P450cam but not CYP101D1. A series of mutagenic, crystallographic, kinetic, and molecular dynamics studies indicate that this mutation locks P450cam into a closed, inactive conformation. In CYP101D1, the D259E mutant changes the rate-limiting step to reduction of the P450-oxy complex, thus opening a window into the critical proton-coupled electron transfer step in P450 catalysis.

Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation. It has been hypothesized that the Pdx-induced shift toward the open state frees the essential Asp251 from salt bridges with Arg186 and Lys178 so that Asp251 can participate in a proton relay network required for O2 activation. This in part explains why P450cam has such a strict requirement for Pdx. One problem with this view is that looser substrate-protein interactions in the open state may not be compatible with the observed regio- and stereoselective hydroxylation. In the present study, molecular dynamics simulations show that Pdx binding favors a conformation that stabilizes the active site and decreases camphor mobility yet retains a partially open conformation compatible with the required proton relay network. The R186A mutant which frees Asp251 in the absence of Pdx retains good enzyme activity, and the crystal structure shows that product, 5-exo-hydroxycamphor, is bound. This indicates that rupture of the Asp251-Arg186 relaxes selectivity with respect to source of electrons and enables X-ray generated reducing equivalents to support substrate hydroxylation. These combined computational and experimental results are consistent with the proposed role of Pdx in assisting the release of Asp251 from ion pairs so that it can participate in proton-coupled electron transfer.

The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein-protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis.

The bacterial cytochrome P450cam catalyzes the oxidation of camphor to 5-exo-hydroxycamphor as the first step in the oxidative assimilation of camphor as a carbon/energy source. CYP101D1 is another bacterial P450 that catalyzes the same reaction. A third P450 (P450tcu) has recently been discovered that has ≈86% sequence identity to P450cam as well as very similar enzymatic properties. P450tcu, however, exhibits three unusual features not found in P450cam. First, we observe product in at least two orientations in the X-ray structure that indicates that, unlike the case for P450cam, X-ray-generated reducing equivalents can drive substrate hydroxylation in crystallo. We postulate, on the basis of molecular dynamics simulations, that greater flexibility in P450tcu enables easier access of protons to the active site and, together with X-ray driven reduction, results in O2 activation and substrate hydroxylation. Second, the characteristic low-spin to high-spin transition when camphor binds occurs immediately with P450cam but is very slow in P450tcu. Third, isothermal titration calorimetry shows that in P450cam substrate binding is entropically driven with a ΔH of >0 while in P450tcu with a ΔH of <0 with a more modest change in -TΔS. These results indicate that despite nearly identical structures and enzymatic properties, these two P450s exhibit quite different properties most likely related to differences in conformational dynamics.

The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron-sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis. We have further examined the differences between these two P450s by determining the effect of spin equilibrium, redox properties, and stability of oxygen complexes. We find that Arx shifts the spin state equilibrium toward high-spin, which is the opposite of the effect of Pdx on P450cam. In both P450s, redox partner binding destabilizes the oxy-P450 complex but this effect is much weaker with CYP101D1. In addition, resonance Raman data show that structural perturbations observed in P450cam upon addition of Pdx are absent in CYP101D1. These data indicate that Arx does not play the same effector role in catalysis as Pdx does with P450cam. The most relevant structural difference between these two P450s centers on a catalytically important Asp residue required for proton-coupled electron transfer. We postulate that with P450cam larger Pdx-assisted motions are required to free this Asp for catalysis while the smaller number of restrictions in CYP101D1 precludes the need for redox partner-assisted structural changes.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hIDO1, the indoleamine group of the substrate Trp is H-bonded to S167 through a bridging water, while that in hTDO is directly H-bonded to H76. Here we show that Trp binding to the Si site or the mutation of S167 to histidine in hIDO1 retards its turnover activity and that the inhibited activity can be rescued by an effector, 3-indole ethanol (IDE). Kinetic studies reveal that the inhibited activity introduced by Trp binding to the Si site is a result of retarded recombination of the ferryl moiety with Trp epoxide to form NFK and that IDE reverses the effect by preventing Trp from binding to the Si site. In contrast, the abolished activity induced by the S167H mutation is primarily a result of ∼5000-fold reduction in the O2 binding rate constant, possibly due to the blockage of a ligand delivery tunnel, and that IDE binding to the Si site reverses the effect by reopening the tunnel. The data offer new insights into structure-based design of hIDO1-selective inhibitors.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.