The bacterial SoxAX cytochromes

Abstract

SoxAX cytochromes are heme-thiolate proteins that play a key role in bacterial thiosulfate oxidation, where they initiate the reaction cycle of a multi-enzyme complex by catalyzing the attachment of sulfur substrates such as thiosulfate to a conserved cysteine present in a carrier protein. SoxAX proteins have a wide phylogenetic distribution and form a family with at least three distinct types of SoxAX protein. The types of SoxAX cytochromes differ in terms of the number of heme groups present in the proteins (there are diheme and triheme versions) as well as in their subunit structure. While two of the SoxAX protein types are heterodimers, the third group contains an additional subunit, SoxK, that stabilizes the complex of the SoxA and SoxX proteins. Crystal structures are available for representatives of the two heterodimeric SoxAX protein types and both of these have shown that the cysteine ligand to the SoxA active site heme carries a modification to a cysteine persulfide that implicates this ligand in catalysis. EPR studies of SoxAX proteins have also revealed a high complexity of heme dependent signals associated with this active site heme; however, the exact mechanism of catalysis is still unclear at present, as is the exact number and types of redox centres involved in the reaction.

Fig. 1

Schematic representation of the Sox pathway reaction cycle with different sulfur substrates, a thiosulfate as substrate, b sulfide as substrate, c sulfite as substrate

Fig. 2

Schematic representation of the reaction cycle of Sox proteins involved in thiosulfate oxidation via the Dsr/Sox pathway

Fig. 3

Phylogenetic relationship of SoxA proteins. Two hundred sixteen SoxA amino acid sequences were analyzed using Mega 5.0. The tree shown was generated using the Neighbor-joining algorithm (Poisson model; uniform rate of evolution for all sites; gap treatment: pairwise-deletion; robustness testing: bootstrap method with 500 resampling cycles). The different types of SoxAX proteins are indicated by black bars and labels TI-TIV. A group of several SoxA related cytochromes was used as the outgroup

Fig. 4

Comparison of the SoxA and SoxX structures from Starkeya novella and Rhodovulum sulfidophilum. a The structure of SnSoxA. The heme cofactor (heme 2, using RsSoxA nomenclature) is highlighted in orange and the Cys A74 and Cys A110 Sγ atoms, which participate in the disulfide linkage, which replaces the heme 1 site, are shown as yellow spheres. b The structure of RsSoxA. Heme cofactors 1 and 2 are highlighted in orange. The N-terminal extension (residues 1–51), which ‘caps’ the heme 2 binding site is represented in dark blue. For both structures, the loop (residues 73–83, SnSoxA; residues 79–87, RsSoxA) which shows a different conformation in the SnSoxA and RsSoxA structures is represented in pink. c The structure of SnSoxX. The N-terminal extension present in the SnSoxX structure is represented in blue and the disulfide linkage, between residues Cys B64 and Cys B175 is shown as yellow spheres (the positions of the Cys Sγ atoms are represented). d The structure of RsSoxX. For both c and d, the heme cofactors and their axial Met and His ligands are shown in orange. Features, which show different conformations in the two structures are highlighted in pink (residues B109–B121 for SnSoxX and residues B97–B119 for RsSoxX)

Fig. 5

Active sites of the SnSoxAX and RsSoxAX structures. Panels a and b SnSoxAX with and without surface representations, respectively; Panels c and d RsSoxAX. In all panels the heme 2 cofactors is represented in orange and the Cys active site residue in hot pink. In the SnSoxAX and RsSoxAX structures, residues Gln 197 and Asp 192, respectively (indicated in stick representations) ‘gate’ access to the active site

Fig. 6

Dimeric structures of the SnSoxAX and RsSoxAX structures. a SnSoxAX; b RsSoxAX. For both structures, the SoxA subunit is shown as a surface representation in light blue. The SoxX subunit is shown as a cartoon in light green and the SoxX heme in orange. c and d Electrostatic surfaces of the SnSoxAX dimer. Regions of positive charge are coloured blue and regions of negative charge, red. Hydrophobic surfaces are represented in white. The heme cofactors are represented in orange. Panel d is related to c by a 90° rotation about the x axis

Fig. 7

EPR spectra of the Type II SoxAX protein from Starkeya novella Panel a CW-EPR spectrum of S. novella SoxAX (50 mM, in 20 mM Tris–HCl, pH 8.0, T = 2 K), Panel b S. novella SoxAX CW-EPR signals in the g ~ 2 region (50 mM, in 20 mM Tris–HCl, pH 8.0, T = 2 K), a experimental spectrum, b,c,d simulations of LS1A, LS1B and LS2, respectively (adapted from [53]) Panel c Spectrum i, experimental CW-EPR spectrum of Cu(II)-loaded S. novella SoxAX, (T = 60.0 K), ii spectrum i corrected for heme dependent components (adapted from [50]