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KMID : 0545120100200030532
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 3 p.532 ~ p.541
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Biochemical and Cellular Investigation of Vitreoscilla Hemoglobin (VHb) Variants Possessing Efficient Peroxidase Activity
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Isarankura Na Ayudhya Chartchalerm
Tansila Natta
Worachartcheewan Apilak
Bulow Leif
Prachayasittikul Virapong
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Abstract
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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 (kcat) 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 H2O2 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 λsoret peak at ~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 Fe2+-ferric uptake regulator protein (Fe2+-Fur), an inducer of FeSOD expression.
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KEYWORD
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Vitreoscilla hemoglobin, Peroxidase-like activity, Enzyme kinetics, Catalytic constant, Proteomic analysis, Iron-superoxide dismutase
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