CRYSTAL-STRUCTURE OF THE DI-HEME CYTOCHROME-C PEROXIDASE FROM PSEUDOMONAS-AERUGINOSA

Background: Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP ) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is locate d in the bacterial periplasm where its likely function is to provide p rotection against toxic peroxides. The soluble 323-residue single poly peptide chain contains two covalent c-type haems with very different p roperties: one of them is a low-potential (-330 mV) centre where hydro gen peroxide is reduced (the peroxidatic site); the other is a high-po tential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azuri n.Results: The crystal structure of the oxidized form of PsCCP has bee n determined to 2.4 ii resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two doma ins, both of them containing a covalent c-haem in a structure reminisc ent of class 1 cytochromes c. The domains are related by a quasi-twofo ld axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands. Conclusions: The likely function df the calcium site is to maintain the structural integrity of the en zyme and/or to modulate electron transfer between the two haem domains . The low-potential haem has two histidine axial ligands (His55 and Hi s71) and the high-potential haem is ligated by His201 and Met275. Ther e are no polar residues at the peroxidatic site in the inactive oxidiz ed enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce new re sidues into the haem pocket.