A recombinant diheme SoxAX cytochrome - implications for the relationship between EPR signals and modified heme-ligands.
The multiheme SoxAX proteins are notable for their unusual heme ligation (His/Cys-persulfide in the SoxA subunit) and the complexity of their EPR spectra. The diheme SoxAX protein from Starkeya novella has been expressed using Rhodobacter capsulatus as a host expression system. rSoxAX was correctly formed in the periplasm of the host and contained heme c in similar amounts as the native SoxAX. ESI-MS showed that the full length rSoxA, in spite of never having undergone catalytic turnover, existed in several forms, with the two major forms having masses of 28687 +/- 4 and 28718 +/- 4 Da. The latter form exceeds the expected mass of rSoxA by 31 +/- 4 Da, a mass close to that of a sulfur atom and indicating that a fraction of the recombinant protein contains a cysteine persulfide modification. EPR spectra of rSoxAX contained all four heme-dependent EPR signals (LS1a, LS1b, LS2, LS3) found in the native SoxAX proteins isolated from bacteria grown under sulfur chemolithotrophic conditions. Exposure of the recombinant SoxAX to different sulfur compounds lead to changes in the SoxA mass profile as determined by ESI while maintaining a fully oxidized SoxAX visible spectrum. Thiosulfate, the proposed SoxAX substrate, did not cause any mass changes while after exposure to dimethylsulfoxide a +112 +/- 4 Da form of SoxA became dominant in the mass spectrum.
Kappler
Ulrike
U
Centre for Metals in Biology, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, Australia. u.kappler@uq.edu.au
Hanson
Graeme R
GR
Jones
Alun
A
McEwan
Alastair G
AG
D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D041921:Hyphomicrobiaceae / Q000201:enzymology / Q000235:genetics; D008024:Ligands; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D010088:Oxidoreductases; C090099:diheme cytochrome c