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Protein Engineering, Vol. 14, No. 6, 415-419, June 2001
© 2001 Oxford University Press

How does heme axial ligand deletion affect the structure and the function of cytochrome b562?

Noriho Kamiya1, Yuko Okimoto1, Zhen Ding1, Hiroko Ohtomo1, Masafumi Shimizu1, Atsushi Kitayama1, Hisayuki Morii2 and Teruyuki Nagamune1,3

1 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7–3–1 Hongo, Bunkyo-ku,Tokyo 113-8656, Japan and 2 National Institute of Bioscience and Human-Technology NIBH-E6, Tsukuba, Ibaraki 305-8566, Japan

We have recently generated a new mutant of cytochrome b562 (cytb562) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb562). The M7A cytb562 can bind heme and the UV-visible absorption spectrum is of a typical high-spin ferric heme. To investigate the effect of the lack of Met7 ligation on the structural integrity of cytb562, thermal transition analyses of M7A cytb562 were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb562 is comparable to the apoprotein despite the presence of heme. On the other hand, exogenous ligands such as cyanide and azide ions are readily bound to the heme iron, indicating that the axial coordination site is available for substrate binding. The peroxidase activity of this mutant is thus examined to evaluate new enzymatic function at this site and M7A cytb562 was found to catalyze an oxidation reaction of aromatic substrates with hydrogen peroxide. These observations demonstrate that the Met7/His102 bis-ligation to the heme iron is crucial for the stable folding of cytb562, whereas the functional conversion of cytb562 is successfully achieved by the loose folding together with the open coordination site.


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