A cambialistic superoxide dismutase in the thermophilic photosynthetic bacterium Chloroflexus aurantiacus

Abstract

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.

FIG. 1.

SDS-polyacrylamide gel electrophoresis and Western blotting of purified recombinant C. aurantiacus SOD. (A) The protein fraction was isolated from E. coli ER2508 expressing C. aurantiacus SOD as the MBP-SOD fusion protein. MBP-SOD was isolated by using an amylose column. Lane 1, protein ladder; lane 2, E. coli whole-cell extract with MBP-SOD; lane 3, amylose column flowthrough; lane 4, pure MBP-SOD eluted from the amylose column. (B) Pure SOD was obtained by factor Xa cleavage of MBP-SOD and was isolated by IEF. Lane 1, protein ladder; lane 2, pure recombinant SOD. (C) Western blot with anti-wtSOD antibodies. Lane 1, prestained protein ladder; lane 2, C. aurantiacus whole-cell extract; lane 3, pure recombinant SOD.

FIG. 2.

Mass spectrum of SOD and MBP cleaved by factor Xa. The SOD peak is at 23,153.01 Da, and there is a second peak at 22,963.33 Da. This second peak is most likely a secondary cleavage product produced by factor Xa. MBP is at 42,491.32 Da, with the doubly charged peak at 21,236.06 Da.

FIG. 3.

Deduced amino acid sequence of C. aurantiacus SOD. This sequence is available from GenBank under accession number AY289213. The N-terminal factor Xa cleavage site and putative secondary structure regions are indicated, and metal-coordinating residues are indicated by boldface type.

FIG. 4.

Neighbor-joining tree of experimentally characterized SODs from bacteria and archaea. The GenBank accession numbers for the protein sequences used are AAL26890, A38461, P00448, CAA44556, CAA11227, P09738, AAC64207, AAA72217, NP_341862, AAF36989, BAA00489, 1BT8_B, P17670, CAA50266, AAA91964, and NP_743076.

FIG. 5.

EPR spectra of MBP-SOD isolated from E. coli grown under two conditions. (A and B) Normal LB growth medium, with production of native MBP-SOD (A) and acidified MBP-SOD (B). (C and D) LB medium supplemented with 1 mM MnSO₄, with production of native MBP-SOD (C) and acidified MBP-SOD (D). Iron and, to a lesser extent, manganese were present in MBP-SOD, and manganese was the only metal detected when E. coli was grown in the presence of 1 mM MnSO₄.

FIG. 6.

Native polyacrylamide gel electrophoresis activity assays of pure recombinant SOD and the effects of H₂O₂ exposure. SOD samples were electrophoresed on polyacrylamide gels under native conditions. The gels were identical except for pretreatment with H₂O₂. Lane 1, 20 μg of E. coli MnSOD; lane 2, 25 μg of E. coli FeSOD; lane 3, 5.4 μg of recombinant C. aurantiacus SOD. (A) Buffer control; (B) pretreatment with 10 mM H₂O₂; (C) pretreatment with 20 mM H₂O₂; (D) pretreatment with 80 mM H₂O₂.