Biochemical and cellular investigation of Vitreoscilla hemoglobin (VHb) variants possessing efficient peroxidase activity.
Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in distal pocket of peroxidases have successfully been introduced into that of the VHb. Fifteen-fold increase in catalytic constant (k(cat)) was obtained in P54R variant, which was presumably attributable to the lower rigidity and higher hydrophilicity of distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either H(2)O(2) or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring T29H mutation apparently demonstrated the spectral shift in both ferric and ferrous forms (406-408 to 411 nm and 432 to 424-425 nm, respectively). All VHb proteins in ferrous state had lambda(soret) peak at approximately 419 nm following the carbon monoxide (CO) binding. Expression of P54R mutant mediated the down-regulation of iron superoxide dismutase (FeSOD) as identified by 2-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived H2O2, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of Fe(2+)-ferric uptake regulator protein (Fe(2+)-Fur), an inducer of FeSOD expression.
Isarankura-Na-Ayudhya
Chartchalerm
C
Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand.
Tansila
Natta
N
Worachartcheewan
Apilak
A
Bülow
Leif
L
Prachayasittikul
Virapong
V
D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052160:Benzothiazoles / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013451:Sulfonic Acids / Q000378:metabolism; D054793:Truncated Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020589:Vitreoscilla / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D052160:Benzothiazoles; D011994:Recombinant Proteins; D013451:Sulfonic Acids; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006639:Histidine; D001120:Arginine; D006861:Hydrogen Peroxide; D010544:Peroxidases