Cytochrome c551 from Starkeya novella

Abstract

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulfidophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 ؎ 10.3 Da and 20,258.6 ؎ 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: ؉133 mV (pH 6.0); ؉104 mV (pH 7.0); ؉49 (pH 7.9) and ؉10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA-and soxX-related gene sequences indicates a parallel development of SoxA and SoxX, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Insights into Structure and Function of the Active Site of SoxAX Cytochromes

“…Mass fingerprints of proteins separated on SDS-polyacrylamide gels were prepared as in Ref. 7 and analyzed using a VoyagerSTR MALDI-TOF mass spectrometer (Applied Biosystems). Electrospray mass spectrometry was performed on a Q-Star mass spectrometer (Applied Biosystems) essentially as in Ref.…”

“…It has been suggested that this modification is responsible for the multiple EPR active states observed for the SoxA active site heme (7,(12)(13)(14).…”

“…The single heme SoxX subunits of group2 SoxAX proteins have a molecular mass of ϳ22-26 kDa (group 1 and 3; ϳ15-16 kDa) (6,7), and the amino acid sequence similarity between these types of SoxX subunits is only 30 -35%. To date, no crystal structures for the group 2 and group 3 SoxAX proteins have been reported despite the obvious differences in protein structure and the potential mechanistic implications.…”

“…The two groups of heterodimeric SoxAX cytochromes can be distinguished based on the number of heme groups present in the SoxA subunit; while group 1 proteins have two heme groups/SoxA subunit (8,9), in group 2 enzymes, only the heme group located close to the proposed SoxAX active site is present (7). The single heme SoxX subunits of group2 SoxAX proteins have a molecular mass of ϳ22-26 kDa (group 1 and 3; ϳ15-16 kDa) (6,7), and the amino acid sequence similarity between these types of SoxX subunits is only 30 -35%.…”

“…SoxAX cytochromes are members of the rare heme-thiolate proteins (5) and can be classified into two groups of heterodimeric and one group of heterotrimeric proteins (6,7), all of which contain the two heme-bearing subunits, SoxA and SoxX (7). In addition, the heterotrimeric SoxAX cytochromes contain another subunit, SoxK, that stabilizes the SoxAX complex (6).…”