ENGINEERING, EXPRESSION AND BIOCHEMICAL-CHARACTERIZATION OF THE HEMOGLOBIN DOMAIN OF A ERWINIA-CHRYSANTHEMI FLAVOHEMOPROTEIN

An artificial hemoglobin-like domain has been constructed by engineeri ng the gene coding for the multi-domain flavohemoprotein from the bact erium Erwinia chrysanthemi. This domain was designed by molecular mode lling, cloned and over-expressed in Escherichia coli. The hole-protein was obtained in large quantities after extraction from inclusion bodi es and refolding in presence of alkaline hemin. The purified 140-resid ue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein. The structural and functional features of this domain in solution were studied using far-ultraviolet circular dichroism, resonance Raman, proton-NMR spectroscopy, flash laser phot olysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin-like domain is able to bin d oxygen and carbon monoxide with very high affinity. It exhibits a ra pid autooxidation which may explain its tight association with a flavi n containing reductase domain. A functional model of this hemoglobin i s discussed and compared with the X-ray structures of other hemoprotei ns.