As shown here, oxidation of the cholesterol side-chain by CYP125 leads to the formation not only of the C26-acid, as previously reported,19
but also of five additional products (M1
) resulting from C-C bond cleavage. The formation of these additional products was uncovered by varying the incubation conditions and carefully analyzing the product HPLC peaks. The identities of the metabolites were established by comparison with authentic standards or by mass spectrometry in combination with deuterium labeling and the use of 18
in the oxidation reaction. The mechanisms proposed for formation of the C-C bond cleavage products () are fully consistent with the deuterium and oxygen labeling results and with the general mechanisms proposed for such reactions.29-31,43
None of the reactions leading to the observed products is entirely unprecedented, but the co-occurrence of this diversity of products in the catalytic turnover of a single substrate by a P450 enzyme is highly unusual. This product diversity has provided us with a tool with which to examine the effect of electron donation from the proximal ligand on formation of the Compound I species.
The role of Compound I in monooxygenation reactions is well documented. Similarly, growing evidence points to the ferric-peroxyanion intermediate as the active oxidant in C-C bond cleavage reactions. All this evidence clearly supports the role of the ferric-peroxy anion in the generation of deformylation products. Thus, the feasibility of the reaction is established by non-enzymatic deformylation of the 19-aldehyde of androstenedione by a porphyrin ferric-peroxy anion model complex.44
Trapping of the ferric-peroxy anion in CYP19 using cryo-freeze quench EPR methods confirms the biological existence of this intermediate and bolsters its potential role in deformylation reactions.45
More directly, the use of radiolabeled substrates and 18
has clearly established the involvement of the ferric-peroxy anion in the formation of acyl-carbon bond cleavage products by CYP5131
results that specifically rule out the participation of a Compound I intermediate. Furthermore, consistent with nucleophilic attack of the peroxy anion on the aldehyde, a distal H-bonding mutant in CYP2B4 that blocks the protonation of the peroxy anion, generated more deformylated metabolites relative to the Compound I-mediated monooxygenation product.46
The peroxy anion mechanism was recently supported by a detailed theoretical analysis.39
Finally, in the present case, the total number of deuterium atoms retained by the metabolites after the oxidation of cholesterol with the heptadeuterated sidechain, excludes alternative mechanisms involving a Compound I intermediate. The branchpoint intermediate in generating the acid and all the C-C bond cleavage products is the C26 aldehyde (). Based on literature precedent, it is likely that the 26-aldehyde acts as the common precursor of all the end products since the same products, and similar product distributions, are obtained when the aldehyde is provided as a substrate (Table S2
) as when the aldehyde is generated internally from cholesterol or 26-hydroxy cholesterol through initial oxidative turnovers. In this system the formation of products from the Compound I-mediated monooxygenation and peroxyhemiacetal-mediated deformylation pathways is controlled by the intrinsic reactivity of the ferric-peroxo anion intermediate. To investigate the effect of increased electron donation from the proximal ligand on partitioning of the aldehyde intermediate into the two reaction pathways, we expressed CYP125 with a selenocysteine replacing the cysteine that provides the proximal thiolate iron ligand.
The introduction of selenocysteine (SeCys) as the proximal iron ligand in a cytochrome P450 enzyme has recently been reported.40,41
Unlike other proximal ligand substitutions, the SeCys proteins retain spectroscopic and catalytic properties comparable to those of their cysteine counterparts, although increased electron donation by the selenolate versus thiolate ligand does cause some differences. These include a shift to longer wavelengths of the Soret maxima of the Fe2+
-CO complexes and a lowering of the redox potentials of the enzymes.34,41
In agreement with these earlier findings, the Soret maximum of the SeCys-substituted CYP125 utilized in these studies was at 459 nm rather than the 450 nm found for the WT* protein. The position of this Soret indicated a high degree of incorporation of SeCys into the protein, an inference supported by the near absence of EPR resonances from the WT* enzyme in the EPR spectrum of SeCYP125 (). Furthermore, comparison of the resonance Raman spectra of the ferric and ferric-nitrosyl hemes in WT* and SeCYP125 proteins show that the SeCys substitution increases electron donation to the metal iron.
More importantly, SeCYP125 binds cholest-4-en-3-one with a similar affinity to that of the WT* enzyme and oxidizes this substrate to the same products, albeit not in exactly the same ratios. When the reaction is run with cholesterol 26-aldehyde as the substrate, both enzymes consume the substrate to essentially the same degree after 60 min, so the differences in product ratios are not due to differential extents of substrate utilization. This result also argues that factors such as differential uncoupling of the two enzymes is not a factor in determining the differences in the product ratios, as higher uncoupling would lead to slower consumption of the aldehyde. It should also be emphasized that the ratio of the products starting with the aldehyde is the most relevant to the conclusions of this paper, as it directly compares the reactions at the branchpoint without any confounding factors that might arise from a requirement for multiple turnovers prior to generating the aldehyde. With both enzymes, all the C-C bond cleavage products are formed, but the 26-acid metabolite is the major product. The primary finding is that the SeCYP125-mediated oxidation produces more cholesterol 26-acid relative to the sum of the C-C bond cleavage products than the WT*. The 1.3 fold increase in the ratio of the 26-acid to deformylation products indicates that selenocysteine substitution favors the formation of Compound I. The oxidation of cholesterol and 26-hydroxycholesterol, the two precursors of the aldehyde, by SeCYP125 also yielded an ~ 1.3 fold increase, relative to the WT*, in the ratio of the acid to homolysis products (). Finally, oxidation of 25,26,26,26,27,27,27-D7-cholesterol by the WT* enzyme was examined. The results show that even with deuterated cholesterol, the amount of acid produced is enhanced relative to the sum of deformylation products. This result argues against any mechanism in which both carboxylic acid formation and decarboxylation are mediated by the ferryl species.
Although the differences in the product ratios obtained with the WT* and SeCYP125 enzymes are essentially identical among the cholesterol derivatives, the product ratios for the same enzyme varied significantly for the different substrates. The steady-state amounts of the cholesterol 26-aldehyde remaining at the end of the 60 min incubation of WT* enzyme with cholesterol and 26-hydroxy cholesterol were calculated to be ~ 5 μM and 2.8 μM, respectively. Similarly for SeCYP125, the amounts of cholesterol 26-aldehyde remaining were ~ 5.8 and 3.1 μM. Although more aldehyde reacted when 26-hydroxy cholesterol was the substrate, overall the amounts of aldehyde reacted with WT* and SeCYP125 at the 60 min time point were comparable.
The ratio of the products differed when the substrate was changed from cholesterol or its 26-oxidized products to cholest-4-en-3-one. This finding is not surprising, as there is no reason to expect that the 3-keto-26-aldehyde generated from cholest-4-en-3-one will bind in the active site and will interact with the protein residues in exactly the same manner as the 3-hydroxy-26-aldehyde of cholesterol. Nevertheless, it is clear that with this substrate the carboxyl-forming branch of the reaction is also favored by selenolate ligation to the iron. More surprising is the finding that the ratio of products exhibited some variation for a given enzyme when comparing the degradation of cholesterol, 26-hydroxycholesterol, and the cholesterol 26-aldehyde. A constant ratio would be expected if no factors intervened other than the intrinsic partitioning of the product pathways at the aldehyde stage. There is no evidence for allosteric effects of cholesterol or its oxidation products on CYP125 catalysis. It is possible that this variation results from a channeling effect that occurs when the substrate is cholesterol or 26-hydroxycholesterol. As shown by the UV-vis binding experiments, a water molecule is displaced from the heme iron on binding of the substrate. This water molecule may slightly perturb the hydrogen bonding pattern in the active site during catalytic turnover, but more importantly, this perturbation may not be the same when a substrate such as cholesterol binds to the enzyme two catalytic turnovers prior to the one that controls the product ratio. Of course, this argument only applies if, at least to some extent, the substrate is processed through multiple turnovers without dissociating from the protein. Interestingly, channeling in the oxidation of aldehydes generated in situ
from precursor molecules has been reported in the reactions of CYP2A6.47
Partitioning of the ferric-peroxo anion intermediate between conversion to Compound I (leading to acid formation) and addition to the aldehyde to give a peroxyhemiacetal (leading to C-C bond cleavage) hinges on the extent to which the intermediate is present as the peroxy anion versus the hydroperoxide. Increased electron donation from the proximal thiolate to the iron bound dioxygen is proposed to increase the pKa
of Compound 0.27
Computational studies suggest that the more electron-donating selenolate proximal ligand should enhance the rate of formation of Compound I.48
In our studies, an increase in the pKa
of the peroxy anion would result in a shift from the anionic towards the hydroperoxo form, and this shift would result in increased formation of Compound I and decreased formation of the peroxyhemiacetal intermediate. This predicts an increased oxidation of the aldehyde to the carboxylic acid at the expense of the deformylation products, as observed here. To our knowledge, this is the first direct evidence emphasizing the role of increased electron donation from the proximal ligand leading to enhanced electron density on the distal oxygen of the ferric-peroxo anion intermediate.
Protonation of the ferric-peroxo anion depends, of course, on the availability of a suitable proton donor on the distal side of the heme. Thus, a number of studies have shown that replacement of a highly conserved distal threonine thought to be involved in this process leads to decreased catalysis and increased uncoupling.46,49
However, in the present instance, the distal Thr272 and its environment are unchanged and cannot be the cause of the difference in protonation of the ferric-peroxo anion.
The change of 1.2-1.3 fold in the ratio of the acid to deformylation products is significant but modest. Importantly, identical ratios are reproducibly observed with both cholesterol and cholest-4-en-3-one, as well as when the 26-alcohol and 26-aldehyde derivatives are used instead of the parent steroids as the substrates. It is difficult to predict the change in the pKa value of the distal oxygen of the ferric-peroxo anion intermediate when a selenolate replaces the thiolate ligand, but the change is not likely to be large. Thus, the expected changes in product ratios should be modest, as observed. Moreover, as the increased electron density on the peroxy anion increases its nucleophilic character, it may promote its addition to the aldehyde and lower the acid to deformylation product ratios.
The extensive partitioning between monooxygenation and deformylation of the C26 aldehyde seen in the oxidation of cholesterol by CYP125 is unique among the known steroid C26-monooxygenases, as CYP27A1, CYP124 and CYP142 produce the alcohol, aldehyde, and acid metabolites without detectably forming the deformylation products.16,17,19,32,50
Thus, the ferric-peroxo anion intermediate in these latter enzymes is either protonated much more quickly than in CYP125, or its possible addition to the aldehyde carbonyl group is suppressed within the active site.