A SINGLE MUTATION OF Leptospira interrogans HEME OXYGENASE GREATLY IMPAIRS ENZYME ACTIVITY

SOLDANO A; CATALANO-DUPUY DL; CECCARELLI EA

MAR DEL PLATA

Congreso; SAIB - 51 Annual Meeting Argentine Society for Biochemistry and Molecular Biology / LI Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2015

monoxide and iron for subsequent use by the pathogen. Heme degradation is a complex process that requires O2 and reducing equivalents. In this study we analyzed the role of phenylalanine-157, a residue located in a loop at 16.5 Åfrom the heme-binding site that is conserved in almost all HOs. It participates in an H-bond network that may deliver H+ and propagates conformational changes to the active site. The wild type enzyme (HOwt) and a F157I mutant werecharacterized by optical absorption spectroscopy. We found that HOwt catalyzed the NADPH/ferredoxin-NADP+ reductase dependent oxidation of heme to biliverdin, whereas the F157I reaction was arrested at verdoheme and did not proceed to biliverdin. There were no differences in the formation of the deoxyferrous complex under anaerobic conditions. However, exposing this intermediate to O2 showed that the oxy-form of F157 was very susceptible to autoxidation in contrast to the stability observed in the HOwt. Furthermore, F157I has low efficiency to hydroxylate heme in the presence of H2O2. The present data point out the importance of F-157 in maintaining the appropriate environment for the HO reaction, the mutation probably produces steric effects that trigger a flexibility attenuation of the heme pocket affecting HO activity.