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Volume 156, Issue 8

Spectroscopic properties of rubber oxygenase RoxA from sp., a new type of dihaem dioxygenase Free

Abstract

Natural rubber [poly-(-1,4-isoprene)] is cleaved to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) by rubber oxygenase A (RoxA) isolated from sp. RoxA has two -type haem centres that show two distinct -bands at 549 and 553 nm in the dithionite-reduced state. A well-resolved midpoint potential ( ′) of –65 mV was determined for one haem by spectrophotometric titrations in the absence of dioxygen with dithionite and ferricyanide as reductant and oxidant, respectively. The midpoint potential of the second haem was not resolvable ( ′ about −130 to –160 mV). One of the two haems was reduced by NADH (549 nm -band), similar to bacterial dihaem peroxidases. Evidence for an electron transfer between the two haems was provided by slow reduction of the second haem (553 nm -band) upon incubation of the partially reduced enzyme at room temperature. Addition of imidazole or related compounds to RoxA led to UV/vis spectral features similar to those observed for partially reduced RoxA. Notably, reduction of RoxA with dithionite or NADH, or binding of compounds such as imidazole, resulted in a reversible inactivation of the enzyme, unlike dihaem peroxidases. In line with this result, RoxA did not show any peroxidase activity. EPR spectra of RoxA as isolated showed two low-spin Fe(III) haem centres, with apparent -values of 3.39, 3.09, 2.23, 1.92 and 1.50. A weak signal in the =6 region resulting from a high-spin Fe(III) haem was also observed with a preparation-dependent intensity that disappeared in the presence of imidazole. Attempts to provide spectroscopic evidence for binding of the natural substrate (polyisoprene latex) to RoxA failed. However, experimental data are presented that RoxA is able to subtract redox equivalents from its substrate or from model compounds. In conclusion, RoxA is a novel type of dihaem dioxygenase with features clearly different from classical cytochrome peroxidases.

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Keyword(s): CCP, cytochrome c peroxidase , HRP, horseradish peroxidase , ICP-MS, inductively coupled plasma MS , NR, natural rubber , ODTD, 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al and PMS, phenazine methosulfate
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2010-08-01
2024-04-25
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Spectroscopic properties of rubber oxygenase RoxA from Xanthomonas sp., a new type of dihaem dioxygenase
Microbiology 156, 2537 (2010); https://doi.org/10.1099/mic.0.038992-0
/content/journal/micro/10.1099/mic.0.038992-0
/content/journal/micro/10.1099/mic.0.038992-0
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