We investigated the physical properties of SeCYP119 to determine to what extent they are altered by replacement of the proximal cysteine thiolate by a larger, more electron donating, ligand. CYP119, a thermophilic P450, is remarkably more stable than mesophilic P450 enzymes. Replacement of the active site cysteine by a selenocysteine did not alter the thermal stability of the enzyme. Some of the thermal stability of CYP119 is due to stacking interactions of aromatic residues at the protein surface (35
), but the insensitivity of the thermal stability to the selenolate for thiolate substitution is surprising. Similarly, the MCD spectra of the ferric and ferrous states of WT CYP119 and SeCYP119 exhibit no significant differences, confirming our previous previous conclusion from EPR and resonance Raman data that the electronic nature at the heme center in the seleno protein is very similar to that of the WT enzyme (16
). However, the redox potential of −265 mV for SeCYP119 is approximately 51 mV lower than that of WT CYP119, in close accord with the redox potential difference of 48 mV between CYP101A1 and SeCYP101A1 (17
). A second phase observed in the redox potential titration with a potential of −67 mV cannot be unambiguously assigned. This potential corresponds to a less electron donating proximal ligand and could possibly reflect the presence of some oxidized selenolate ligand. Thus, although the structural properties of CYP119 are minimally perturbed by the selenocysteine replacement, it causes a substantial change in the redox potential.
In view of the change in redox potential, the role of the electron rich selenocysteine ligand in the kinetics of Cpd I formation was investigated. Formation of Cpd I has been characterized by SVD analysis of the oxidation of WT CYP119 by m
). Oxidation of WT CYP119 with sub-stoichiometric amounts of m
CPBA generated a Cpd I intermediate with absorption maxima at 370, 610 and 690 nm (3
). The Cpd I spectrum was calculated from SVD analysis of the rapid scan data in which the protein underwent little reaction under limiting peracid conditions (22
). In our hands, oxidation of WT CYP119 with stoichiometric and sub-stoichiometric concentrations of m
CPBA produced an intermediate in the SVD analysis with spectral characteristics similar to those of the previously reported Cpd I (3
). Interestingly, oxidation of SeCYP119 under similar conditions did not yield a detectable Cpd I. The overall change in absorbance in the reaction of SeCYP119 with m
CPBA was very small compared to the reaction of WT CYP119 under the same conditions and the SVD analysis of the data using various kinetic models failed to generate a characteristic Cpd I intermediate. This suggests three possibilities: a)
SeCYP119 does not react with m
the reaction occurs but Cpd I is not formed, or c)
Cpd I is formed but is very quickly reduced to the ferric state. While SeCYP119 reacted to about a three-fold lesser extent (~6%) compared to the WT enzyme (~17%), as judged by the overall change in the absorbance with 1 equivalent of m
CPBA, the amount of SeCYP119 that reacted should suffice to observe Cpd I in the SVD analysis. Furthermore, the fact that SeCYP119 catalyzes lauric acid hydroxylation in the presence of excess H2
) or m
CPBA clearly suggests the formation of a Cpd I intermediate. Thus, we infer that the reaction of SeCYP119 with m
CPBA yields an intermediate, presumably a seleno Cpd I, that is too rapidly reduced to be detected. Based on the SVD analysis, the apparent rate constant for the reaction of m
CPBA with SeCYP119 was estimated to be 3.4 × 106
. However, this value has to be interpreted carefully as it may involve multiple reactions.1
More importantly, the estimated rate constant (kapp
) represents the slower step in the kinetics and, as Cpd I was not observed, this is presumably the initial reaction of m
CPBA with SeCYP119. Our data indicate that m
CPBA reacts with SeCYP119 at a rate comparable to that of the WT enzyme.
With low concentrations of the oxidant, it is possible that Cpd I decayed faster than it was formed, precluding its accumulation and spectroscopic observation. We therefore increased the rate of formation of Cpd I by increasing the concentration of the oxidant. Although these conditions cause some heme destruction, excess m
CPBA has been used before in the oxidation of CYP101A1 to detect the formation of a Cpd I like species (36
). However, Cpd I was not detected in the SVD analysis when SeCYP119 was oxidized with five- or ten-equivalent excesses of m
CPBA. Our various attempts to detect the formation of Cpd I, including a)
oxidation at higher pH (pH 7.0 and 7.8), b)
using peroxyacetic acid as an alternate oxidizing agent, and c)
oxidation in the presence of lauric acid failed to yield a detectable Cpd I intermediate. Taken together, these results argue for the formation in SeCYP119 of a Cpd I intermediate that decays faster than it is formed. It is likely that the electron-rich selenolate ligand reduces Cpd I as it is formed.
Reaction of SeCYP119 with excess mCPBA generated a species with a Soret maximum at 406 nm (P406) that is ~ 10 nm blue-shifted from the absorption maximum of the native ferric protein. Surprisingly, only 40% of the heme was lost during the oxidation of SeCYP119. Thus, SeCYP119 is much more resistant to heme destruction than the WT protein. The active site of SeCYP119 has clearly undergone an unprecedented oxidative modification that effectively competes with the heme destruction pathway. The nature of this P406 species is discussed below.
An intermediate with an absorption maximum at 406 nm has been encountered in the m
CPBA-mediated oxidation of CYP101A1 (37
), and more recently CYP153A6 from Mycobacterium
sp. HXN-1500 (39
). This intermediate was assigned to Cpd ES with a neutral (or protonated) Fe(IV)=O species and a radical located on a nearby tyrosine or tryptophan residue (40
). Importantly, Cpd ES is relatively rapidly reduced to the native ferric state in a process that is accelerated by mild reducing agents such as ascorbic acid or guaiacol. In contrast, addition of ascorbic acid (1 mM final concentration) to the SeCYP119 P406 species did not alter the absorbance spectrum. Thus, although the UV-vis spectral properties of SeCYP119 P406 are similar to those observed in the reaction of m
CPBA with CYP101A1, its stability to mild reducing agents clearly differentiates it from a Cpd ES.
Oxidation of SeCYP119 with peroxynitrite (PN) generated a Cpd II like intermediate (neutral Fe(IV)=O species) similar to that of the WT enzyme. SeCYP119 reacts more slowly with PN than does WT CYP119, but the resulting Cpd II is more stable. Importantly, Cpd II decayed back to the native ferric protein without forming a P406 intermediate, presumably because Cpd II is not as highly oxidizing as Cpd I. The P406 intermediate thus derives from a reaction intermediate other than Cpd II.
Reduction of SeCYP119 P406 with dithionite in the presence of carbon monoxide immediately yielded a 422 nm peak in the difference spectrum that subsequently underwent complete, time-dependent conversion over 5–10 min to an Fe(II)-CO spectrum with a maximum at 456 nm identical to that of the original SeCYP119. These changes indicate that the structural features that cause the 406 nm maximum are eliminated upon reduction with sodium dithionite. Based on these results, we speculate that P406 has a six-coordinated low spin ferric heme with an oxidatively modified proximal selenocysteine ligand that is reduced by dithionite back to selenocysteine. Consistent with this hypothesis, DTT, a thiol based reducing agent, reduced the chromophore in P406 to the native ferric heme with its normal coordination and its signature α and β bands. Treatment with DTT apparently reduced the oxidatively modified proximal SeCys without reducing the heme iron atom. Restoration of the native protein by DTT was supported by an LC/MS/MS spectrum of the trypsin digested P406 species that revealed an alkylated SeCys active site peptide identical to that of SeCYP119 not exposed to mCPBA. Taken together, these results strongly argue that P406 is a pure species containing an oxidatively modified active site that is completely reduced by treatment with dithionite or DTT to regenerate the native SeCYP119 protein.
It is tempting from our findings to propose oxidation of the SeCys to the selenenic acid (Se-OH), as this oxidation would be reversed by reducing agents such as dithionite and DTT. Our results rule out the presence of irreversibly oxidized forms of the proximal SeCys ligand such as SeO2
H and SeO3
H, as these should not be reduced by DTT and dithionite. Our attempts to trap a selenenic acid species using 4-chlor-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) have been unsuccessful. The proximal SeCys is deeply buried in the proximal side of the active site and is not readily accessible to NBD. However, we cannot rule out an intra-molecular protein cross-link to form something like a selenenyl amide with the neighboring lysine residue. Thus, an alternate pathway is proposed that involves oxidation of the proximal SeCys to generate a stable two electron oxidized species that competes with heme destruction (). The exact mechanism for this transformation is not clear at the moment. Selenols (pKa ~ 5.2) are more acidic than thiols (pKa ~ 8.7) and hence are deprotonated under physiological conditions (42
). In addition, the one electron reduction potential of SeCys is approximately 0.5 V lower than that of cysteine (43
) and hence SeCys should reduce Cpd I more easily than the cysteine thiolate.
Proposed pathway for the reaction of SeCYP119 with mCPBA to generate the P406 species
Strikingly, while weaker proximal electron donors like histidine fail to support the basic spectral and catalytic properties of a P450 enzyme, a stronger electron donating selenium ligand maintains the spectroscopic characteristics but yields a less active enzyme (15
). The two-fold lesser specific activity of SeCYP119 may reflect oxidation of the selenolate ligand. MCD studies indicate that the hydrogen bonding network in the proximal cysteine pocket of CYP101A1 protects the cysteine ligand from oxidation (45
). The more acidic selenolate in SeCYP119 may disrupt this hydrogen bonding network, or simply as a result of its more oxidizable nature may not be efficiently protected against oxidation by the active site environment.
In conclusion, SeCYP119 has comparable physical properties to WT CYP119. However, the mCPBA-mediated shunt pathway does not generate a detectable Cpd I under conditions that allowed its observation with the WT protein, probably because the Cpd I intermediate is rapidly reduced by the selenolate ligand. Interestingly, oxidation with excess mCPBA produced a stable species with an absorption maximum at 406 nm that was (a) unable to catalyze substrate oxidation when exposed to peroxides and lauric acid, (b) resistant to heme destruction, (c) unable to interact with azoles, (d) reduced to the normal, unmodified ferrous state by dithionite, and (e) reduced to the normal, unmodified ferric state by DTT. P406 is thus a novel, reversible, intermediate so far unique to SeCYP119 that may involve two electron oxidation of the proximal selenolate ligand. The results also establish the remarkable extent to which the active site and chemistry of P450 enzymes is uniquely attuned to the thiolate ligand, with ligands of higher or lower electron donating ability impairing function.