Cytoglobin (Cgb) represents a fourth member of the globin superfamily in mammals, but its function is unknown. Site-directed mutagenesis, in which six histidine residues were replaced with alanine, was carried out, and the results indicate that the imidazoles of His81 (E7) and His113 (F8) bind to the heme iron as axial ligands in the hexacoordinate and the low-spin state. The optical absorption, resonance Raman, and IR spectral results are consistent with this conclusion. The redox potential measurements revealed an E' of 20 mV (vs NHE) in the ferric/ferrous couple, indicating that the imidazole ligands of His81 and His113 are electronically neutral. On the basis of the nu(Fe-CO) and nu(C-O) values in the resonance Raman and infrared spectra of the ferrous-CO complexes of Cgb and its mutants, it was found that CO binds to the ferrous iron after the His81 imidazole is dissociated, and three conformers are present in the resultant CO coordination structure. Two are in closed conformations of the heme pocket, in which the bound CO ligand interacts with the dissociated His81 imidazole, while the third is in an open conformation. The nu(Fe-O2) in the resonance Raman spectra of oxy Cgb can be observed at 572 cm(-1), suggesting a polar heme environment. These structural properties of the heme pocket of Cgb are discussed with respect to its proposed in vivo oxygen storage function.

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Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R-CH‫؍‬N-OH) to their corresponding nitrile (R-C'N). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 Å resolution and the n-butyraldoxime-and propionaldoxime-bound OxdREs at 1.8 and 1.6 Å resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe 3؉ heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.