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 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.
Chu
G C
GC
Department of Biochemistry, the Central Laboratory for Research and Education, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
Katakura
K
K
Tomita
T
T
Zhang
X
X
Sun
D
D
Sato
M
M
Sasahara
M
M
Kayama
T
T
Ikeda-Saito
M
M
Yoshida
T
T
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001205:Ascorbic Acid / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D003353:Corynebacterium diphtheriae / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D006418:Heme; D006639:Histidine; D006419:Heme Oxygenase (Decyclizing); C104107:HmuO protein, bacteria; D000409:Alanine; D001205:Ascorbic Acid