A) Experiments to prove the proposed reduction mechanism for the formation of borneol (borneol cycle):
The proposed reduction mechanism (Fig. 3.7) suggests that water present in the active site of the enzyme is responsible for formation of borneol and H2O2. The isotope
labeled experiments (Fig. 3.2) and the steady-state kinetics (Fig. 3.6) in H2O and D2O
were performed to verify borneol formation. The formation of hydrogen peroxide was verified by 17O NMR. The hypothesis shown in Fig. 3.7 involves compound I as an important intermediate in the borneol cycle. Although this mechanism is supported by NMR and kinetics experiments, a few more experiments can be planned for future studies.
i) Characterisation of compounds I and II, that form in the borneol cycle:
The first question that needs to be addressed is how one could verify if borneol cycle occurs in the active site of the enzyme. This question can’t be answered by just the formation of products: borneol and H2O2. The involvement of compound I and compound
II-H in the mechanism has not been shown experimentally, and observing these reactive intermediates would provide more evidence for the proposed mechanism.
The initial attempts to verify the shunt pathway was performed by Egawa et.al. 275 in which the substrate free P450cam and m-CPBA were mixed and rapid-scan stopped-
flow studies were performed. The weak band at 694 nm and a blue-shifted Soret peak at 367 nm were assigned to a porphyrin π-cation radical. Recent attempts to characterize compound I by stopped-flow kinetics were performed by Green et.al. 46 In their experiments, the compound I was characterized 35 ms after mixing the ferric CYP119 (a P450 isolated from Sulfolobus solfataricus
)
and m-CPBA at 4 °C.reactions differ in the reaction of compound I: for hydroxylation compound I abstracts a H atom from the substrate, giving an alkyl radical and compound II-H. The hydroxylation product then forms by a rebound mechanism, in which the OH radical (effecively coordinated to Fe in compound II-H) joins with the carbon radical to furnish the alcohol. In the borneol cycle, compound I abstracts a H atom from water and then proceeds through the cycle as described in Chapter 3 (Fig. 3.7). The hydroxylation reaction was monitored by stopped-flow kinetics. 79,276 Similar experiments can also be performed for borneol cycle to characterize the compound I and compound II by UV-Visible spectroscopy (stopped flow kinetics) and elucidate the low O2 cycle mechanism. The
main difficulty for this experiment includes the capturing of compound I and II-H intermediates as the lifespan of compound I is expected to be 35-100 ms.
ii) EPR experiments to identify the radical intermediates that form in borneol cycle: The EPR characterization of compound-ES (Fig. 1.2) was performed by Schünemann et.al. 277 In their experiment, the reaction mixture containing the substrate- free P450cam and m-CPBA were freeze-quenched at -110 °C after a reaction time of 8
ms. Green et.al. have also reported the EPR measurements of compound I for CYP119 (a P450 isolated from Sulfolobus solfataricus). 278,46 For this, the reaction mixture containing CYP119 and m-CPBA were freeze-quenched ˂25K and the formation of a new paramagnetic radical was assigned to compound I.
Similar experiments can also be planned with freeze-quenched P450cam, m-
CPBA and camphor to check for the formation of compounds I and II. One difficulty could be the overlapping signals between protein radical, compound I and m- chlorobenzoic radical and their assignments.
iii) Monitoring of borneol cycle by UV-Visible spectroscopy:
The reaction between m-CPBA and the resting P450 forms compound I, and its formation can be monitored by UV-Visible spectroscopy or by extraction and
may cause difficulty in monitoring the reaction by UV spectroscopy. Monitoring by BSTFA derivatization and GC-MS will show the presence or absence of m-chlorobenzoic acid, provided the acid can be extracted quantitatively from the aqueous buffer.
iv) Energy-calculations of the intermediates of borneol cycle:
The thermodynamical calculations of the intermediates of borneol cycle (shown in Fig. 3.8) are estimates only. The energy estimates can be known better if the intermediates of the borneol cycle (Fig. 3.7) are simulated, using validated ab initio protocols. For now, only rough estimates were provided from the electrochemical data
279 and Gaussian calculations.
B) Endosulfan dehalogenation:
Phthaldialdehyde, formed in the biodegradation process, was characterized by
1H and 13C NMR. The phenolic intermediate (23) was also characterized recently. Still,
additional experiments need to be performed to confirm that the phthaldialdehyde formed was indeed derived from endsulfan diol.
i) If the phenolic intermediate (23) is indeed an intermediate during the biodegradation process, enzymatic assays can be performed with the mutant IND1 under shunt conditions to verify if it and phthaldialdehyde form (also see v below). If the experiment is run in D2O, and 23 froms from 19 (Figs. 4.9 and 4.10) and
phthaldialdehyde (25) forms trom 23 by a reduction akin to the borneol cycle described in Chapter 3, then compound 23 should be deuterated at the 4-position and phthaldialdehyde should be dideuteriated at the 4 and 5 positions (see v below).
ii) The release of CO2 in the biodegradation can be verified by 13C studies by
labeling endosulfan dialdehyde. The Schematic representation of the reaction is shown below.
Cl Cl Cl Cl Cl Cl O O dialdehyde [O] P450 mutant HO Cl O O Cl O O HO labeled CO2 7
.
.
The 5-13C labeled hexachlorocyclopentadiene, which would be required to synthesize 7-13C endosulfan dialdehyde, is not available commercially. Therefore, this approach may not work due to practical difficulties.
iii) To check the formation of phthaldialdehydes from endosulfan diol, an experiment with 13C labelled endosulfan diol can be planned. The (4+2) cyclization reaction of hexachlorocyclopentadiene and 2,3-13C maleic acid should give endosulfan diacid. Reduction of endosulfan diacid with LiAlH4 can furnish the desired diol.
Cl Cl Cl Cl Cl Cl OHOH 5 6 5,6 13C endosulfan diol P450cam mutants O O R1 R2 R1 = OH or H R2 = OH or H 13C labelled phthaldialdehydes ?
iv) To verify that all the chlorines are indeed eliminated as Cl-, detailed mass distribution experiments need to be done. In 35Cl NMR, it is possible to quantify the amount of chloride present in the solution. Therefore, it should be possible to verify that 6 Cl- are released from every endosulfan diol used and/or for every phthaldialdehyde produced. For such an assay to work, however, it is essential that all three phthaldialdehydes (22, 23 and 25) be quantitatively extracted from the aqueous assay mixture.
v) To verify that water is indeed the source of H-atoms added to o-quinone 19, the endosulfan diol dehalogenation reaction needs to be done in D2O, under Ar sparge
could be done with the P450 mutant, PdX, PdR and NADD. If the “borneol cycle” is active, then we do not expect to see deuteriated phthaldialdehyde in these controls.
Cl Cl Cl Cl Cl Cl OH OH 5 6 P450cam mutants O O R1 R2 R1 = D R2 = D D2O buffer Ar sparge ?
vi) Monitoring of the biodegradation reaction: If the UV/visible peak absorbances of the intermediates (23 and 25) are known and if distinct, then this reaction can also be studied by stopped-flow kinetics.
vii) To further verify the mechanism of endosulfan dehalogenation, steady-state kinetics need to be done in normal and D2O buffers, under O2 pressure and under Ar
pressure, just as was done in Chapter 3. This will help us check for O2 dependence of
the reduction steps and provide indirect evidence that a process similar to the borneol cycle occurs in the dehalogenation mutants as well.