Spectroscopic properties of rubber oxygenase RoxA from Xanthomonas sp., a new type of dihaem dioxygenase
- 1 August 2010
- journal article
- Published by Microbiology Society in Microbiology
- Vol. 156 (8), 2537-2548
- https://doi.org/10.1099/mic.0.038992-0
Abstract
Natural rubber [poly-(cis-1,4-isoprene)] is cleaved to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) by rubber oxygenase A (RoxA) isolated fromXanthomonassp. RoxA has twoc-type haem centres that show two distinctα-bands at 549 and 553 nm in the dithionite-reduced state. A well-resolved midpoint potential (E0′) 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 (E0′ about −130 to –160 mV). One of the two haems was reduced by NADH (549 nmα-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 nmα-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 apparentg-values of 3.39, 3.09, 2.23, 1.92 and 1.50. A weak signal in theg=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 cytochromecperoxidases.Keywords
This publication has 43 references indexed in Scilit:
- Activation and Catalysis of the Di-Heme Cytochrome c Peroxidase from Paracoccus pantotrophusStructure, 2006
- Structural and Biochemical Characterization of DHC2, a Novel Diheme CytochromecfromGeobacter sulfurreducens,Biochemistry, 2005
- O2- and α-Ketoglutarate-Dependent Tyrosyl Radical Formation in TauD, an α-Keto Acid-Dependent Non-Heme Iron DioxygenaseBiochemistry, 2003
- Crystal Structure of Nitrosomonas europaea Cytochrome c Peroxidase and the Structural Basis for Ligand Switching in Bacterial Di-heme PeroxidasesBiochemistry, 2001
- Gordonia polyisoprenivorans sp. nov., a rubber-degrading actinomycete isolated from an automobile tyreInternational Journal of Systematic and Evolutionary Microbiology, 1999
- Overproduction of theBradyrhizobium japonicum c-Type Cytochrome Subunits of thecbb3Oxidase inEscherichia coliBiochemical and Biophysical Research Communications, 1998
- Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding siteFEMS Microbiology Letters, 1996
- Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosaStructure, 1995
- Properties and function of the two hemes in Pseudomonas cytochrome c peroxidaseBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1983
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976