A single mutation converts Alr5027 from cyanobacteria Nostoc sp. PCC 7120 to a heme-binding protein with heme-degrading ability.

HutZ from Vibrio cholerae (VcHutZ) is a dimeric protein that catalyzes oxygen-dependent degradation of heme. The reaction mechanism is the same as that of canonical heme oxygenase (HO), but the structure of HutZ is quite different from that of HO. Thus, we postulate that HutZ has evolved via a different pathway from that of HO. The Alr5027 protein from cyanobacteria possessing proteins potentially related to ancestral proteins utilizing O in enzymatic reactions is homologous to HutZ family proteins (67% similarity), but the heme axial ligand of HutZ is not conserved in Alr5027. To investigate whether Alr5027 can bind and degrade heme, we expressed Alr5027 in Escherichia coli and purified it. Although Alr5027 did not bind heme, replacement of Lys164, corresponding to the heme axial ligand of HutZ, with histidine conferred heme-binding capability. The K164H mutant produced verdoheme in the reaction with HO, indicating acquisition of heme-degradation ability. Among the mutants, the K164H mutant produced verdoheme most efficiently. Although the K164H mutant did not degrade heme through ascorbic acid, biliverdin, the final product of VcHutZ, was formed by treatment of verdoheme with ascorbic acid. An analysis of Trp103 fluorescence indicated elongation of the distance between protomers in this mutant compared with VcHutZ-the probable cause of the inefficiency of ascorbic acid-supported heme-degradation activity. Collectively, our findings indicate that a single lysine-to-histidine mutation converted Alr5027 to a heme-binding protein that can form verdoheme through HO, suggesting that HutZ family proteins have acquired the heme-degradation function through molecular evolution from an ancestor protein of Alr5027.