Effects of mutations in active site heme ligands on the spectroscopic and catalytic properties of SoxAX cytochromes.
By attaching a sulfur substrate to a conserved cysteine of the SoxYZ carrier protein SoxAX cytochromes initiate the reaction cycle of the Sox (sulfur oxidation) multienzyme complex, which is the major pathway for microbial reoxidation of sulfur compounds in the environment. Despite their important role in this process, the reaction mechanism of the SoxAX cytochromes has not been fully elucidated. Here we report the effects of several active site mutations on the spectroscopic and enzymatic properties of the type II SoxAX protein from Starkeya novella, which in addition to two heme groups also contains a Cu redox centre. All substituted proteins contained these redox centres except for His231Ala which was unable to bind Cu(II). Substitution of the SoxA active site heme cysteine ligand with histidine resulted in increased microheterogeneity around the SoxA heme as determined by CW-EPR, while a SnSoxAXC236Asubstituted protein revealed a completely new, nitrogenous SoxA heme ligand. The same novel ligand was present in SnSoxAXH231ACW-EPR spectra, the first time that a ligand switch of the SoxA heme involving a nearby amino acid has been demonstrated. Kinetically, SnSoxAXC236Aand SnSoxAXC236Hshowed reduced turnover, and in assays containing SoxYZ these mutants retained only ~25% of the wildtype activity. Together, these data indicate that the Cu redox centre can mediate a low level of activity, and that a possible ligand switch can occur during catalysis. It also appears that the SoxA heme cysteine ligand (and possibly the low redox potential) is important for an efficient reaction with SnSoxYZ/thiosulfate.
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia.
James R
JR
Kilmartin
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia.
Paul V
PV
Bernhardt
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia.
Rabeb
R
Dhouib
Centre for Advanced Imaging, The University of Queensland, Brisbane, Qld 4072, Australia.
Graeme R
GR
Hanson
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia.
Mark J
MJ
Riley
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Qld 4072, Australia. Electronic address: u.kappler@uq.edu.au.
Ulrike
U
Kappler
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009154:Mutation; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012230:Rhizobiaceae / Q000201:enzymology* / Q000235:genetics; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013885:Thiosulfates / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D003580:Cytochromes; D008024:Ligands; D011994:Recombinant Proteins; D013885:Thiosulfates; D006418:Heme; D003300:Copper; D003545:Cysteine
Cytochromes; EPR; Heme thiolate proteins; Redox properties; SoxAX; Thiosulfate oxidation