Asparagine 887 is crucially involved in hydroperoxide rearrangement

It is known that the amide function of an asparagine in the I-helix of hydroperoxy fatty acid rearranging P450s is crucially involved in peroxide cleavage (Chiang et al., 2006; D.-S. Lee et al., 2008). Notably, sequence alignments did not lead to identification of a homologue residue in PpoA and in the initial model of the P450-domain, no asparagine was at the expected position.

However, a neighboring residue within the I-helix is an asparagine (Asn887), but its side chain points by approximately 90° away from the expected direction. Assuming that this detail might be false predicted, a respective variant (Asn887Val) was constructed. Although the conducted variation did not lead to a qualitative change of the product pattern and only the three enzymatic products known from conversions by wild type enzyme, i.e. 8-HODE, 8-HPODE and 5,8-DiHODE, were produced, there was a significant effect on the hydroperoxide isomerase activity causing a decrease in the relative amount of rearranged product and a concomitant accumulation of DOX-derived products (Figure 26).

During characterization of the purified PpoA-variant, it became obvious that the conducted mutation leads additionally to a reduced soret absorption of the enzyme. To address whether this reduced vis-absorption is caused by a lower heme-content of either the DOX-or the P450-domain, or merely reflects altered cofactor coordination in one or the other P450-domain, Dr. Alistair Fielding (MPI for Biophysical Chemistry, Goettingen) recorded cw X-band EPR-spectra of the variant as well as of wild type enzyme. The obtained data reveal that there is no difference in the high-spin heme indicating that the cofactor of the DOX-domain is not affected by the conducted variation. In contrast, the low-spin signal assigned as P450-heme (Fielding et al., 2011) was not only reduced by approximately 40 %, but also the tensor was altered (Figure 27). Since the g-values of a P450 enzyme are very sensitive to the cofactor’s ligation sphere, these changes might indicate an altered coordination as one would expect, if the amide of Asn887 forms a hydrogen bonding network involving the subtrate’s peroxide and the heme-iron.

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Figure 26: The Asn887Val-variant of PpoA exhibits a significantly reduced hydroperoxy fatty acid isomerase activity as compared to the wild type. This effect is revealed by a decreased amount of rearranged product and a concomitant accumulation of DOX-derived products. The reaction of the respective variant with linoleic acid was extracted after 2 min and product patterns in these extracts were measured by LC-MS². Shown are the arithmetic means of products quantified for three independent conversions as well as the corresponding standard deviation.

Figure 27: cw X-EPR spectra of the low-spin heme species in PpoA-Asn887Val (75 µM;

green) as compared to wild type (75 µM;

black). Qualitative differences in g₁ are marked. Additionally, the heme occupancy in the variant is only about 60 % of the wild type value. Spectra were recorded by Dr.

Alistair Fielding with details specified in literature (Fielding et al., 2011). The spectrum of the variant was measured for a single preparation and the wild type spectrum is consistent with spectra obtained for several other preparations of this enzyme 0

10 20 30 40 50 60 70 80 90 100

5.8-DiHODE 8-HODE 8-HPODE

relative amount of formed product [%]

wild type

Asn887Val

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A final experiment to shed light on the role of asparagine 887 was based on the idea that the asparagine might structurally resemble the functional homologue position in class III P450s. As it was shown for PpoA (Brodhun et al., 2009), this P450 class in general exhibits only weak and transient binding of typical P450-heme ligands like CO and imidazole (Yeh et al., 2005; D.-S. Lee et al., 2008). Taken crystallographic data into account, this observation is explained by the carboxamide of the catalytic asparagine sterically hindering the direct coordination of small ligands to the heme iron (D.-S. Lee et al., 2008). To probe a similar position of Asn887 directly above the distal heme plane in PpoA’s P450-domain, an imidazole titration was performed as already outlined in section 4.2.5 and figure 14. Briefly, wild type enzyme and the Asn887Val-variant were both titrated with imidazole and the red-shift of the soret band was monitored.

Interestingly, titrations of both enzymes yielded basically the same dissociation constant (kD 6 to 11 µM, assuming a one-site binding model), indicating that Val still might be too bulky to permit access to the heme or that the asparagine in PpoA indeed is farer apart from the heme than in other class III P450s, as it was predicted in the structural model (Section 4.3.1.2). Notably, the measured affinity is roughly 50-times higher as it was reported for prostacyclin synthase (Yeh et al., 2005), which might additionally point out a more accessible heme. By imidazole titration of PpoA’s His1004Ala-variant, in which the P450-heme is absent (Brodhun et al., 2009), it could be shown that also the histidine coordinated heme of the DOX-domain is binding imidazole. Thus, for proper evaluation of the spectral binding assays, consideration of a two-site binding model was necessary. Therefore the binding parameters obtained for titration of the His1004Ala-variant (kD 34µM) were defined as (fixed) parameters of imidazole binding to the DOX-domain and affinity parameters of imidazole binding to the P450-heme were subsequently calculated from titrations of wild type enzyme and the Asn887Val-variant, respectively, with fixed parameters for imidazole binding to the DOX-heme and the imidazole binding parameters for the P450-heme fitted to a two-site binding model. For both, the wild type enzyme and its Asn887Val-variant, the thus obtained KD did not differ significantly and were in the range of 80 µM. This value still represents an about 6-fold higher affinity of imidazole to the P450-heme of PpoA as compared to prostacyclin synthase. Again the missing influence of a less bulky residue at position 887 on ligand access supports the idea that this position in PpoA is less important in shielding of the P450 heme as in other class III P450s.

Taken together the here presented data substantiate the involvement of Asn887 in hydroperoxide rearrangement and justify the refinement of the initially obtained predicted structure. Nevertheless, the heme-binding of imidazole suggests that the P450-heme of PpoA is shielded to a lesser extent than in other class III P450s. This in turn might indicate that the catalytic competent asparagine, which is responsible for the steric shielding, has a different position and is in agreement with the failure to identify a catalytic competent asparagine by sequence alignments.

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