Posttranslational Modification of Heme b in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases including myeloperoxidase (MPO) and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. The present study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A and D109A/E238A which could be recombinantly produced as apo-proteins in E. coli, fully reconstituted to the respective heme b proteins and posttranslationally modified by hydrogen peroxide. This for the first time allows not only to directly compare the catalytic properties of the heme b and PTM forms, but also to study the impact of D109 and E238 on PTM and catalysis including Compound I formation and the two-electron reduction of Compound I by bromide, iodide and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238 - in contrast to exchange of D109 - does not cause significant structural rearrangements and changes of the catalytic properties neither in the heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.