STRUCTURAL AND FUNCTIONAL FEATURES OF PSEUDOMONAS CYTOCHROME-C PEROXIDASE

The secondary structure of Pseudomonas cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) has been predicted from the established amino acid sequence of the enzyme using a Chou-Fasman-type algorithm. The amount of alpha-helicity thus obtained is in agreement with previously obtained results based on circular dichroic measurements at far UV. The two heme c moieties of the enzyme have earlier been shown to have widely different characteristics, e.g., the redox potentials of the hemes differ with about 600 mV, and carry out different functions in the enzyme molecule. The structural comparisons made in this study enlighten the observed functional differences. The first heme in the polypeptide chain, heme 1, has in its environment a folding pattern generally encountered in cytochromes. In the region of the sixth ligand, however, profound differences are noted. The cytochromal methionine has been replaced by a lysine with a concomitant lowering of redox-potential thus making peroxidatic activity possible. Around heme 2, extra amino acid residues have been added to the peroxidase as compared with Rhodospirillum molischianum cytochrome c2 core structure in the 20's loop. After completion of the cytochromal fold around heme 2 an additional tail consisting of 25 residues is linked. This tail shows no stabilizing elements of secondary structure, but contains a strongly hydrophobic segment which suggests a possible membrane contact site of this extrinsic membrane protein. Heme 2 is concluded to have a cytochromal function in the molecule. To further elucidate the functional properties of the enzyme, a noncovalent two-fragment complex was produced by specific cleavage of the peroxidase by Pseudomonas elastase. The complex was studied with respect to its properties to the native enzyme. The two-fragment complex of Pseudomonas peroxidase retains the overall conformation of the native enzyme showing, however, no heme-heme interaction. Thus, a comparison of the properties of the native enzyme with those of the two-fragment complex permitted some conclusions to be drawn on the structure of the enzyme as well as the mechanism of heme-heme interaction. From the present results we conclude that the two distal heme surfaces in the peroxidase are oriented toward each other. This structural arrangement allows an inter-heme communication in the enzyme molecule and it also forms the structural basis for the enzyme mechanism. The structural comparisons also give insight into the evolution of an ancestral cytochrome c into an efficient peroxidase that has a versatile control mechanism in heme-heme interaction.