Histidine 20, the crucial proximal axial heme ligand of bacterial heme oxygenase Hmu O from Corynebacterium diphtheriae |
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Authors: | Chu G C Katakura K Tomita T Zhang X Sun D Sato M Sasahara M Kayama T Ikeda-Saito M Yoshida T |
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Institution: | Department of Biochemistry, the Central Laboratory for Research and Education, Yamagata University School of Medicine, Yamagata 990-9585, Japan. |
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Abstract: | The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His --> Ala Hmu O mutants. Reconstituted with hemin, 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) as the proximal heme ligand in Hmu O, and this is confirmed by resonance Raman results from the ligand-free ferrous heme-H20A. All eight His --> Ala mutants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His(20) is crucial to Hmu O catalysis. Its absence by point mutation has inhibited the conversion of hemin to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonance 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 pentacoordinate CO-bound hemeprotein. The appearance of delta(Fe-C-O) in C. diphtheriae Hmu O H20A but not mammalian HO-1 mutant H25A indicates that the heme environment between the two heme oxygenases is different. |
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