Piotr J. Mak

Resonance Raman Characterization of the Peroxo and Hydroperoxo Intermediates in Cytochrome P450

Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450(cam) (CYP101) are reported. Owing to the fact that proton delivery to the active site is hindered in this mutant, the unprotonated peroxo-ferric intermediate is observed as the primary species after radiolytic reduction of the oxy-complex in frozen solutions at 77 K. In as much as previous EPR and ENDOR studies have shown that annealing of this species to approximately 180 K results in protonation of the distal oxygen atom to form the hydroperoxo intermediate, this system has been exploited to permit direct RR interrogation of the changes in the Fe-O and O-O bonds caused by the reduction and subsequent protonation. Our results show that the nu(O-O) mode decreases from a superoxo-like frequency near approximately 1130 cm(-1) to 792 cm(-1) upon reduction. The latter frequency, as well as its lack of sensitivity to H/D exchange, is consistent with heme-bound peroxide formulation. This species also exhibits a nu(Fe-O) mode, the 553 cm(-1) frequency of which is higher than that observed for the nonreduced oxy P450 precursor (537 cm(-1)), implying a strengthened Fe-O linkage upon reduction. Upon subsequent protonation, the resulting Fe-O-OH fragment exhibits a lowered nu(O-O) mode at 774 cm(-1), whereas the nu(Fe-O) increases to 564 cm(-1). Both modes exhibit a downshift upon H/D exchange, as expected for a hydroperoxo-ferric formulation. These experimental RR data are compared with those previously acquired for the wild-type protein, and the shifts observed upon reduction and subsequent protonation are discussed with reference to theoretical predictions.

Differential hydrogen bonding in human CYP17 dictates hydroxylation versus lyase chemistry.

Raman spectra of oxygenated intermediates of Nanodisc incorporated human CYP17 in the presence of natural substrates directly confirm that substrate structure effectively alters H-bonding interactions with the critical Fe-O-O fragment so as to dictate its predisposition for one of two alternative reaction pathways, providing a realistic structural explanation for substrate control of CYP17 reactivity that has profound physiological implications.

Defining CYP3A4 Structural Responses to Substrate Binding. Raman Spectroscopic Studies of a Nanodisc-Incorporated Mammalian Cytochrome P450

Resonance Raman (RR) spectroscopy is used to help define active site structural responses of nanodisc-incorporated CYP3A4 to the binding of three substrates: bromocriptine (BC), erythromycin (ERY), and testosterone (TST). We demonstrate that nanodisc-incorporated assemblies reveal much more well-defined active site RR spectroscopic responses as compared to those normally obtained with the conventional, detergent-stabilized, sampling strategies. While ERY and BC are known to bind to CYP3A4 with a 1:1 stoichiometry, only the BC induces a substantial conversion from low- to high-spin state, as clearly manifested in the RR spectra acquired herein. The third substrate, TST, displays significant homotropic interactions within CYP3A4, the active site binding up to 3 molecules of this substrate, with the functional properties varying in response to binding of individual substrate molecules. While such behavior seemingly suggests the possibility that each substrate binding event induces functionally important heme structural changes, up to this time spectroscopic evidence for such structural changes has not been available. The current RR spectroscopic studies show clearly that accommodation of different size substrates, and different loading of TST, do not significantly affect the structure of the substrate-bound ferric heme. However, it is here demonstrated that the nature and number of bound substrates do have an extraordinary influence on the conformation of bound exogenous ligands, such as CO or dioxygen and its reduced forms, implying an effective mechanism whereby substrate structure can impact reactivity of intermediates so as to influence function, as reflected in the diverse reactivity of this drug metabolizing cytochrome.

Resonance Raman Spectroscopy of the Oxygenated Intermediates of Human CYP19A1 Implicates a Compound I Intermediate in the Final Lyase Step

CYP19A1, or aromatase, a cytochrome P450 responsible for estrogen biosynthesis in humans, is an important therapeutic target for the treatment of breast cancer. There is still controversy surrounding the identity of reaction intermediate that catalyzes carbon–carbon scission in this key enzyme. Probing the oxy-complexes of CYP19A1 poised for hydroxylase and lyase chemistries using resonance Raman spectroscopy and drawing a comparison with CYP17A1, we have found no significant difference in the frequencies or isotopic shifts for these two steps in CYP19A1. Our experiments implicate the involvement of Compound I in the terminal lyase step of CYP19A1 catalysis.

Unveiling the Crucial Intermediates in Androgen Production

Significance The human enzyme cytochrome P450 17A1 (CYP17A1) catalyzes the critical step in the biosynthesis of the male sex hormones, and, as such, it is a key target for the inhibition of testosterone production that is necessary for the progression of certain cancers. CYP17A1 catalyzes two distinct types of chemical transformations. The first is the hydroxylation of the steroid precursors pregnenolone and progesterone. The second is a different reaction involving carbon–carbon (C-C) bond cleavage, the mechanism of which has been actively debated in the literature. Using a combination of chemical and biophysical methods, we have been able to trap and characterize the active intermediate in this C-C lyase reaction, an important step in the potential design of mechanism-based inhibitors for the treatment of prostate cancers. Ablation of androgen production through surgery is one strategy against prostate cancer, with the current focus placed on pharmaceutical intervention to restrict androgen synthesis selectively, an endeavor that could benefit from the enhanced understanding of enzymatic mechanisms that derives from characterization of key reaction intermediates. The multifunctional cytochrome P450 17A1 (CYP17A1) first catalyzes the typical hydroxylation of its primary substrate, pregnenolone (PREG) and then also orchestrates a remarkable C17–C20 bond cleavage (lyase) reaction, converting the 17-hydroxypregnenolone initial product to dehydroepiandrosterone, a process representing the first committed step in the biosynthesis of androgens. Now, we report the capture and structural characterization of intermediates produced during this lyase step: an initial peroxo-anion intermediate, poised for nucleophilic attack on the C20 position by a substrate-associated H-bond, and the crucial ferric peroxo-hemiacetal intermediate that precedes carbon–carbon (C-C) bond cleavage. These studies provide a rare glimpse at the actual structural determinants of a chemical transformation that carries profound physiological consequences.

Differential Control of Heme Reactivity in Alpha and Beta Subunits of Hemoglobin: A Combined Raman Spectroscopic and Computational Study

The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or β hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe–histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or β chains, prior to the quaternary R–T and T–R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe–His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem.2002, 98, 149). Experimentally, the νFeHis evolution is faster for β than for α chains, and pump–probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 μs for β but not for α hemes, an interval previously shown to be the first step in the R–T transition. In the α chain νFeHis dropped sharply at 20 μs, the final step in the R–T transition. The time courses are fully consistent with recent computational mapping of the R–T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A.2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.

Resonance Raman Studies of Cytochrome P450 2B4 in Its Interactions with Substrates and Redox Partners

Resonance Raman studies of P450 2B4 are reported for the substrate-free form and when bound to the substrates, benzphetamine (BZ) or butylated hydroxytoluene (BHT), the latter representing a substrate capable of inducing an especially effective conversion to the high-spin state. In addition to studies of the ferric resting state, spectra are acquired for the ferrous CO ligated form. Importantly, for the first time, the RR technique is effectively applied to interrogate the changes in active site structure induced by binding of cytochrome P450 reductase (CPR) and Mn(III) cytochrome b 5 (Mn cyt b 5); the manganese derivative of cyt b 5 was employed to avoid spectroscopic interferences. The results, consistent with early work on mammalian P450s, demonstrate that substrate structure has minimal effects on heme structure or the FeCO fragment of the ferrous CO derivatives. Similarly, the data indicate that the protein is flexible and that substrate binding does not exert significant strain on the heme peripheral groups, in contrast to P450 cam, where substantial effects on heme peripheral groups are seen. However, significant differences are observed in the RR spectra of P450 2B4 when bound with the different redox partners, indicating that the heme structure is clearly sensitive to perturbations near the proximal heme binding site. The most substantial changes are displacements of the peripheral vinyl groups toward planarity with the heme macrocycle by cyt b 5 but away from planarity by CPR. These changes can have an impact on heme reduction potential. Most interestingly, these RR results support an earlier observation that the combination of benzphetamine and cyt b 5 binding produce a synergy leading to unique active site structural changes when both are bound.

Experimental documentation of the structural consequences of hydrogen-bonding interactions to the proximal cysteine of a cytochrome P450.

Resonance Raman spectroscopy is used to document, for the first time, a 6 cm−1 decrease of the Fe-S stretch by introducing an H-bond donor into the proximal pocket of a cytochrome P450, which interacts with the key cysteine thiolate axial ligand. The anticipated trans-effect on bound exogenous ligands is also confirmed and evidence is obtained supporting intimate interaction of the new histidyl-imidazole fragment with the heme periphery.

Defining the Structural Consequences of Mechanism-Based Inactivation of Mammalian Cytochrome P450 2B4 Using Resonance Raman Spectroscopy

In view of the potent oxidizing strength of cytochrome P450 intermediates, it is not surprising that certain substrates can give rise to reactive species capable of attacking the heme or critical distal-pocket protein residues to irreversibly modify the enzyme in a process known as mechanism-based (MB) inactivation, a result that can have serious physiological consequences leading to adverse drug-drug interactions and toxicity. While methods exist to document the attachment of these substrate fragments, it is more difficult to gain insight into the structural basis for the altered functional properties of these modified enzymes. In response to this pressing need to better understand MB inhibition, we here report the first application of resonance Raman spectroscopy to study the inactivation of a truncated form of mammalian CYP2B4 by the acetylenic inhibitor 4-(tert-butyl)phenylacetylene, whose activated form is known to attach to the distal-pocket T302 residue of CYP2B4.

Resonance Raman spectroscopy reveals that substrate structure selectively impacts the heme-bound diatomic ligands of CYP17

An important function of steroidogenic cytochromes P450 is the transformation of cholesterol to produce androgens, estrogens, and the corticosteroids. The activities of cytochrome P450c17 (CYP17) are essential in sex hormone biosynthesis, with severe developmental defects being a consequence of deficiency or mutations. The first reaction catalyzed by this multifunctional P450 is the 17α-hydroxylation of pregnenolone (PREG) to 17α-hydroxypregnenolone (17-OH PREG) and progesterone (PROG) to 17α-hydroxyprogesterone (17-OH PROG). The hydroxylated products then either are used for production of corticoids or undergo a second CYP17 catalyzed transformation, representing the first committed step of androgen formation. While the hydroxylation reactions are catalyzed by the well-known Compound I intermediate, the lyase reaction is believed to involve nucleophilic attack of the earlier peroxo- intermediate on the C20-carbonyl. Herein, resonance Raman (rR) spectroscopy reveals that substrate structure does not impact heme structure for this set of physiologically important substrates. On the other hand, rR spectra obtained here for the ferrous CO adducts with these four substrates show that substrates do interact differently with the Fe-C-O fragment, with large differences between the spectra obtained for the samples containing 17-OH PROG and 17-OH PREG, the latter providing evidence for the presence of two Fe-C-O conformers. Collectively, these results demonstrate that individual substrates can differentially impact the disposition of a heme-bound ligand, including dioxygen, altering the reactivity patterns in such a way as to promote preferred chemical conversions, thereby avoiding the profound functional consequences of unwanted side reactions.