EXPRESSION AND CHARACTERIZATION OF TRUNCATED HUMAN HEME OXYGENASE (HHO-1) AND A FUSION PROTEIN OF HHO-1 WITH HUMAN CYTOCHROME-P450 REDUCTASE

A human heme oxygenase (hHO-1) gene without the sequence coding for th e last 23 amino acids has been expressed in Escherichia coli behind th e pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the fi rst time for detailed mechanistic studies. The purified, truncated hHO -1/heme complex is spectroscopically indistinguishable from that of th e rat enzyme and converts heme to biliverdin when reconstituted with r at liver cytochrome P450 reductase. A self-sufficient heme oxygenase s ystem has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for t he 20 amino acid membrane binding domain. Expression of the fusion pro tein in pCWori(+) yields a protein that only requires NADPH for cataly tic turnover. The failure of exogenous cytochrome P450 reductase to st imulate turnover and the insensitivity of the catalytic rate toward ch anges in ionic strength establish that electrons are transferred intra molecularly between the reductase and heme oxygenase domains of the fu sion protein. The V-max for the fusion protein is 2.5 times higher tha n that for the reconstituted system. Therefore, either the covalent te ther does not interfere with normal docking and electron transfer betw een the flavin and heme domains or alternative but equally efficient e lectron transfer pathways are available that do not require specific d ocking.