Cytochrome P450s protect against oxidative damage via a preferred orientation of the heme cofactor

ABSTRACT: Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenases that utilise a cysteine thiolate ligated heme moiety to perform a wide range of demanding oxidative transformations. Given the oxidative power of active intermediate formed within P450s during their active cycle, it is remarkable that these enzymes can avoid self-oxidation as well as retaining the axial cysteine ligand in the deprotonated – and thus highly acidic – thiolate form. Whilst little is known about the process of heme incorporation during P450 folding, there is an overwhelming preference for one heme orientation within the P450 active site. Indeed, very few structures to date contain an alternate heme orientation, of which two are OxyA homologues from glycopeptide antibiotic (GPA) biosynthesis. Given the apparent preference for the unusual heme orientation shown by OxyA enzymes, we investigated the OxyA homologue from kistamicin biosynthesis, which is an atypical GPA. We determined that OxyAkis is highly sensitive to oxidative damage by peroxide, with both UV an EPR measurements shows a rapid bleaching of the heme signal. We determined the structure of OxyAkis and found a mixed population of heme orientations present in this enzyme. Analysis further revealed that an unprecedented oxidative modification of the heme was detected, which that correlated with the presence of the alternate heme orientation in the protein. These results provide evidence that the typical heme orientation in Cytochrome P450s can help to prevent potential autocatalytic modification of the heme – and hence deactivation of the enzyme – during P450 catalysis. It also suggests that some P450 enzymes involved in GPA biosynthesis may be especially prone to oxidative damage due to the heme orientation found in their active sites.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ralf Schittenhelm  

LAB HEAD: Max Cryle

PROVIDER: PXD030867 | Pride | 2022-05-20

REPOSITORIES: Pride