4.5 Reaction of P450 CAM with Peroxynitrite
4.5.1 P450 CAM -catalyzed Nitration of Phenol by Peroxynitrite . 94
P450CAM is probably the most thoroughly investigated P450 enzyme at all. Lots of kinetic, substrate specific, mechanistic and mutagenesis-related studies have been done with this enzyme (for reviews see [247, 248]). According to the known crystal structure and sequence of P450CAM some tyrosine residues are located close to the active site [249, 250]
We previously reported about a significant increase of phenol nitration by PN, in the presence of iron complexes, especially of heme- and heme-thiolate-enzymes [116, 163]. Therefore also a nitration increase in reactions of phenol with PN was expected, when CAM was present at catalytic concentrations . Since not only the camphor-containing but also the -free enzyme was available, we expected a significant effect of an empty or occupied active site on the nitration activity.
Tab.14 shows the yields of nitration, hydroxylation and dimerization prod-ucts from the reaction of phenol with PN in presence and absence of different heme and heme-thiolate proteins. These values were determined for the reaction of 5 mM phenol with 800 µM PN at pH 7 and 7.5, using RP-HPLC. The metallo proteins were present at concentrations of 1-2 µM. As previously shown [116, 163], the nitration increase by 1 µM NOR is much higher compared to that of 2µM HRP. Whereas 1 µM camphor-free CAM could increase the nitration at pH 7 and 7.5 by 80-90 %, 1 µM camphor-containing CAM did not and yielded values comparable to the protein-free control. The hydroxylation at pH 7 was best suppressed by NOR, followed by camphor-free CAM and HRP. Once more camphor-containing CAM showed the smallest effect. More suprising were the results concerning the dimerization: Normally the increase in phenol nitration is accompanied by an increase in dimerization as well, because phenoxyradicals are the common intermediary species in both reactions, the iron-catalyzed
and proton-assisted pathway. As can be seen in the table, dimerization in the HRP-catalyzed reaction is increased by a factor of about 18, in the case of NOR by a factor of 4, with camphor containing CAM it reaches the value of the control and with the camphor-free form it is even decreased by 30 %. It should be noticed that 2,4’-BP is the major dimerization product [130] and was not available as a standard, therefore it was quantified by its relative peak area in HPLC chromatograms. The simultaneous increase in nitration and decrease in dimerization was first observed with this enzyme. Camphor-free CAM seems to be able to increase the nitration by a highly specific mechanism, by which less free radicals are formed or where formed radicals are somehow caged in a more efficient way.
Table 14: CAM catalysis of phenol nitration by PN. 5 mM phenol were reacted with 800µM peroxynitrite in 0.1 M potassium phosphate buffer pH 7 and 7.5 at RT.
4 RESULTS 96 4.5.2 Autocatalyzed Nitration of P450CAM by Peroxynitrite
Since nitration of added phenol is increased, it would be also feasible if the nitration of tyrosyl-residues within the enzyme requires low concentrations of PN. To prove an autocatalytic nitration of CAM we used the Western blot technique and stained with anti-NT as already described for other nitrated P450-enzymes, such as PGI2 synthase, BM3 and NOR [10, 163, 116]. Fig.85 shows an anti-NT Western blot of camphor-containing and -free CAM samples after treatment with different concentrations of PN. Obviously the free-form shows a clear signal at 50 µM, whereas the camphor-containing-form shows an intense signal only at 250 µM.
Figure 85: α-NT Western blot of CAM in the camphor-free and -containing (25 µM camphor) form after treatment with differ-ent concdiffer-entrations of PN.
Figure 86: α-NT Western blot of CAM, nitrated in the presence and absence of 12.5 µM metyrapone.
In another experiment the same samples as taken for these Western blots in Fig.85 were injected to a HPLC-system, optimized for the detection of NT-positive peptides at 428 nm and alkaline pH. Before injection, the enzyme was digested by trypsin. Fig.87 shows the HPLC chromatograms of camphor-free (A) and -containing (B) trypsin-digested CAM after treatment with 50 or 500 µM PN. The same pattern of NT-positive peaks was found in both cases, except a strong signal at 12.3 min, which only appeared in the camphor-free enzyme. The areas of all peaks (except that one of the porphyrin) increased with increasing PN-concentration and reached a plateau at around 500 µM PN (see inserts in Fig.87). It should be noted that the chromatograms are shown at different scales and that the areas of all peptide-containing NT-positive peaks were decreased by a factor of 2-3 in the camphor-containing experiments (see inserts). Thus autocatalytic nitration of CAM was hindered by camphor.
Figure 87: Chromatograms of trypsin-digested CAM (2.5 µM). (A) in the camphor-containing form (25µM camphor) after treatment with 50 (grey, dotted line) and 250 µM (black, solid line) PN at pH 7.5. The insert shows the area of NT-positive peptide-peaks in dependence of PN-concentration (tR=15.8 min, open circles; tR=19.5 min, open triangles;
tR=22 min, open squares, tR=28 min, solid circles). (B) in the camphor-free form after treatment with 50 (grey, dotted line) and 250 µM (black, solid line) PN at pH 7.5 (Insert:
tR=12.3 min, solid squares; the other markers are similar to those in insert A).
The peaks were isolated and send for MS-analysis. The following Tab.15 shows the identified tyrosine-containing peptide fragments and if present, the mass-shift due to nitration.
R.time [min] tR=12.3 tR=15.5 tR=18 tR=19.5 tR=22 tR=28 tR=37 Fragment no peptide 366-372 213-239 179-197 91-109, 300-313 198-211 porphyrin
Nitrated Y - - - 179 96 and 305 201 or 203
-m/z, q 663.5, + 815.49, + 923.16, 3+ 736.33, 3+ 1099.5, 2+; 565.62, 3+ 890.97, 2+
-Table 15: Identified NT-positive fragments from trypsin-digested CAM, after treatment with 250µM PN (R.time=retention time).
For better support of the autocatalytic nitration of CAM we additionally used metyrapone to block the active site. Metyrapone is well known for its tight binding to the heme-iron and an active site located tyrosine in CAM [250, 251].
This inhibitor was already efficient at low concentrations. Fig.86 shows an anti-NT Western blot of CAM after treatment with different concentrations of PN in presence and absence of 12.5 µM metyrapone. Without metyrapone, the nitration of CAM starts at PN concentrations of around 25 µM, whereas in presence of this inhibitor the nitration shows only a faint signal at 50 µM. Also for the higher concentrations of PN the metyrapone-free samples always show a stronger staining compared to the metyrapone-containing one and it clearly turned out, that nitration of the enzyme was decreased by a factor of 2-3 when the active site was blocked. The same effect was observed for the peak area of NT-positive peptides in chromatograms, where the samples used for the Western blot in Fig.86 were injected after digestion with trypsin (see Fig.88). Tab.16
4 RESULTS 98 shows the areas of the NT-positive peptides in absence or presence of 50 µM metyrapone. The areas of the Y179- and Y201/203-containing fragments only are half as high in the metyrapone-containing samples and the peak of the Y96- and Y305-containing fragments disappeared completely. To ensure that metyrapone at the employed concentrations had no effect on the PN-mediated nitration a comparable Western blot was made for BM3 and no difference could be observed between the nitration of metyrapone-containing and -free samples (see Fig.89).
Furthermore metyrapone had no effect on the PN-mediated nitration of phenol, when employed at these concentrations (not shown).
Figure 88: Chromatograms of trypsin-digested CAM (2.5 µM) after treatment with 250 µM PN in presence and absence of 50µM metyrapone at pH 7.
Figure 89: α-NT Western blot of BM3, nitrated in the presence and absence of 12.5 µM metyrapone.
Rel.Area
Sample Y179 Y96+305 Y201/203
without metyrap. 25168 72237 10607
+ 50 µM metyrap. 12895 - 5927
Table 16: Area of NT-positive peaks of digested CAM, nitrated in absence and presence of 50µM metyrapone.
4.5.3 Camphor-binding and CO-binding of Native and PN-treated