How does heme axial ligand deletion affect the structure and the function of cytochrome b(562)?
We have recently generated a new mutant of cytochrome b(562) (cytb(562)) in which Met7, one of the axial heme ligands, is replaced by Ala (M7A cytb(562)). The M7A cytb(562) 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 cytb(562), thermal transition analyses of M7A cytb(562) were conducted. From the thermodynamic parameters obtained, it is concluded that the folding of M7A cytb(562) 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 cytb(562) 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 cytb(562), whereas the functional conversion of cytb(562) is successfully achieved by the loose folding together with the open coordination site.
Kamiya
N
N
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Okimoto
Y
Y
Ding
Z
Z
Ohtomo
H
H
Shimizu
M
M
Kitayama
A
A
Morii
H
H
Nagamune
T
T
D019943:Amino Acid Substitution; D052160:Benzothiazoles; D001665:Binding Sites; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006139:Guaiacol / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013451:Sulfonic Acids / Q000378:metabolism; D013816:Thermodynamics
D052160:Benzothiazoles; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D008024:Ligands; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006418:Heme; D006139:Guaiacol; C017850:cytochrome b562, E coli