Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-α84-cystein-lyase (PVBPEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-α-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate bu er inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km= 12-16μM, depending on [PecA], kcat≈1.2×10-4.s-1) and the apoprotein (Km=2.4μM at 14μM PCB, kcat≈0.8×10-4.s-1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEClyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.