Cross-linking of dicyclotyrosine by the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis proceeds through a catalytic shunt pathway

Abstract

CYP121, the cytochrome P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C-C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C-C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme-substrate complex. This intermediate exhibits a high-spin (S = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical-based mechanisms for the C-C bond coupling reaction.

Keywords: C–C bond coupling; cyclodipeptide; cytochrome P450; diradical; electron transfer; enzyme kinetics; metabolism; oxygen activation; tuberculosis.

Figure 1.

Proposed peroxide shunt pathway of CYP121 reaction within the general P450 mechanism. The shunt pathway directly arrives at the hydroperoxo intermediate without the need for receiving electrons and a proton from an external donor (i.e. NAD(P)H) via a reductase system.

Figure 2.

Spectroscopic characterization of substrate cYY binding to CYP121. All substrate binding experiments were monitored using nearly saturating concentrations of cYY, 400 μm. A, UV-visible spectra of CYP121 (10 μm) incubated with cYY; B, EPR characterization of CYP121 (250 μm; black spectrum) binding to cYY (red spectrum); C, stopped-flow UV-visible spectroscopic monitoring of the kinetics of cYY binding to CYP121 shows complete conversion to the ES complex form within the first 250 ms.

Figure 3.

Kinetic characterization of substrate cYY binding to CYP121. A, single-wavelength stopped-flow data collected at 388 nm monitoring formation of enzyme–substrate complex. Reaction conditions were pH 7.5, 21 °C, 5.05 μm heme after mixing. Residuals from fitting stopped-flow data with single- and double-exponential curves are shown. B, plot of reciprocal relaxation times from double-exponential fitting of single-wavelength stopped-flow data collected at pH 7.5 (5 μm heme after mixing). The slower phase shows saturation behavior at high cYY concentrations. Experiments were limited by low solubility of cYY in water. Error bars originate from fitting S.D. values from fitting multiple experimental data sets. C, replots of cYY concentration dependence data showing the sum and product of two observed reciprocal relaxation times from double-exponential fitting of data.

Figure 4.

Transient kinetic analysis of the ES complex (10 μm CYP121 and 400 μm CYY) reacting with peracetic acid (2 mm). A, full spectra of the first 10 s of reaction showing decay of the ES complex. B, difference spectra showing return back to the resting enzyme within 10 s with multiple clear isosbestic points signifying a clear transition back to the resting state during this time period and showing the development of a 427-nm intermediate between 5 and 449 ms. After this time, the intermediate decayed and shifted to 433 nm. C, single-wavelength kinetic traces monitoring the regeneration of the resting enzyme at the expense of the ES complex. D, plots of substrate binding (described in the legend to Fig. 3) showing the formation of the ES complex.

Figure 5.

Stopped-flow UV-visible kinetic characterization of CYP121 (5 μm) reacting with peracetic acid (2 mm). A, 0–100 ms; B, 100 ms to 2 s; C, 2–20 s. Insets, difference spectra showing three intermediates observed for each transition.

Figure 6.

Mass spectrometry analysis of ferric CYP121-mediated cYY cross-linking using peracetic acid as the oxidant. A, total ion count of enzyme assay mixture; B, mass-spectral detection of cYY (325 m/z) at 8.1 min; C, selected ion monitoring of both cYY and mycocyclosin shows a new peak at 4.8 min after incubation of reaction mixture for 10 min; D, molecular mass detection of the cross-linked mycocyclosin (323 m/z) after incubation of the enzyme, cYY, and peracetic acid.

Figure 7.

EPR analysis of the enzyme–substrate complex (250 μm) reacting with peracetic acid (10 mm). The black trace shows the ES complex of cYY and CYP121. A new high-spin ferric heme intermediate is formed in 5 ms with concomitant decay of the low-spin ES complex (red trace). The EPR spectra also include the new high-spin intermediate at 160 (blue) and 300 ms (green). The final decayed reaction complex after 10 s of reaction is shown by a dashed line, which does not contain the intermediate. EPR spectra were obtained at 5 K, 9.6-GHz microwave frequency, and 1-milliwatt microwave power. mT, milliteslas.

Figure 8.

EPR analysis of the resting CYP121 enzyme (250 μm) reacting with peracetic acid (2 mm). The resting CYP121 enzyme is present as a heterogeneous low-spin ferric heme species (black trace). CYP121 was mixed in a 1:1 ratio and quenched in liquid ethane after reaction times of 100 ms (navy trace), 2 s (blue), and 20 s (cyan). The reaction generated at least one high-spin species at g = 6.67, 5.77, and 2 that increased in intensity after longer reaction times. EPR spectra were obtained at 5 K, 9.6-GHz microwave frequency, and 1-milliwatt microwave power. mT, milliteslas.

Figure 9.

HPLC chromatogram (monitored at 280 nm) showing formation of mycocyclosin from reactions of 5 μm CYP121, 400 μm cYY iodosylbenzene (PhIO, red trace), or 400 μm PAA (blue). The product mycocyclosin elutes at 4.3 min. The PhIO reaction generates more side products than the PAA reaction. For the PhIO reaction, the background at 358 nm was subtracted to correct the baseline. mAU, milliabsorbance units.

Figure 10.

Steady-state kinetic analysis of the peroxidase activity of CYP121 (100 nm) with ABTS (2 mm) and PAA (0.02–5 mm). A, representative spectra of CYP121 peroxidase activity as monitored by UV-visible spectroscopy for 1.5 min. B, EPR spectroscopic detection of ABTS^+·. C, single-wavelength stopped-flow data monitoring formation of ABTS^+· at 420 nm. D, Michaelis–Menten fit to the kinetic data as a function of PAA concentration. AU, absorbance units.

Figure 11.

Proposed mechanistic models for the CYP121-mediated cYY cross-linking reaction using peracetic acid as the oxidant. The top two mechanisms are proposed using compound I as the oxidant (A and B), and the bottom two using the peroxo intermediate as the active oxidant (C and D). In each case, two pathways are proposed for the formation of a second radical and subsequent C–C bond formation.