Histidine 20, the crucial proximal axial heme ligand of bacterial heme oxygenase Hmu O from Corynebacterium diphtheriae

The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Cor ynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histi dines in Hmu O is the proximal heme ligand, we have constructed and express ed the plasmids for eight His --> Ala Hmu O mutants. Reconstituted with hem in, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy . EPR of the NO-bound ferrous heme-Hmu O mutant complexes reveals His(20) a s the proximal heme ligand in Hmu O, and this is confirmed by resonance Ram an results from the ligand-free ferrous heme-H20A All eight His --> Ala mut ants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His(20) is crucial to Hmu O c atalysis. Its absence by point mutation has inhibited the conversion of hem in to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonanc e Raman of the CO-bound ferrous heme-H20A corroborates this and reveals an Fe-C-O bending mode, delta(Fe-C-O), the first reported for a pentacoordinat e GO-bound hemeprotein. The appearance of delta(Fe-C-O) in C. diphtheriae H mu O H20A but not mammalian HO-1 mutant H25A indicates that the heme enviro nment between the two heme oxgygenases is different.