Novel Heme Ligation in a c-type Cytochrome Involved in Thiosulfate Oxidation:  EPR and MCD of SoxAX from Rhodovulum sulfidophilum

Abstract

The SoxAX complex of the bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that plays an essential role in photosynthetic thiosulfate and sulfide oxidation. The three heme sites of SoxAX have been analyzed using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies. Heme-3 in the ferric state is characterized by a Large g(max) EPR signal and has histidine and methionine axial heme iron ligands which are retained on reduction to the ferrous state. Hemes-1 and -2 both have thiolate plus nitrogenous ligand sets in the ferric state and give rise to rhombic EPR spectra. Heme-1, whose ligands derive from cysteinate and histidine residues, remains ferric in the presence of dithionite ion. Ferric heme-2 exists with a preparation-dependent mixture of two different ligand sets, one being cysteinate/histidine, the other an unidentified pair with a weaker crystal-field strength. Upon reduction of the SoxAX complex with dithionite, a change occurs in the ligands of heme-2 in which the thiolate is either protonated or replaced by an unidentified ligand. Sequence analysis places the histidine/methionine-coordinated heme in SoxX and the thiolate-liganded hemes in SoxA. SoxAX is the first naturally occurring c-type cytochrome in which a thiolate-coordinated heme has been identified.

Structural and Functional Characterization of the Unusual Triheme Cytochrome Bound to the Reaction Center of Rhodovulum sulfidophilum

“…It seems likely to us that the g z ϭ 2.63 to g z ϭ 2.47 transition corresponds to a relief of steric constraints on the conformation of the two axial heme ligands, similar to what has been described for His/His-and His/Met-ligated hemes (27,28). In SoxAX, the cysteine ligand of one of the two Cys/His hemes is probably chemically modified (26). The mass spectroscopic analysis excludes this possibility for the triheme cytochrome in the R. sulfidophilum RC (data not shown).…”

“…The present mutagenesis study confirms this hypothesis. A case of Cys/His ligation in the native state of a heme-containing enzyme was recently reported for the SoxAX system involved in thiosulfate oxidation (26). Strikingly, SoxAX was also purified from R. sulfidophilum.…”

Flavocytochrome P450 BM3 Mutant A264E Undergoes Substrate-dependent Formation of a Novel Heme Iron Ligand Set

“…Cysteinate-ligated heme enzymes include the Cys-aqua-ligated cytochrome P450 enzymes and the nitric-oxide synthases (see Refs. 45 and 49), and the Cys-Hisligated heme in the SoxAX protein from Rhodovulum sulfidophilum, with a role in thiosulfate oxidation (62). However, the Cys-Glu ligation observed in the A264E mutant of P450 BM3 is an unprecedented heme iron ligand set.…”

“…Changing water for carboxylate at the distal side of the heme results in only very minor band shifts. For imidazole-bound P450, native CooA, and the hemes in subunit I of SoxAX, all of which have a nitrogenous ligand distal to cysteinate, the CT transitions are at 1180 nm (45), 1120 nm (60), and 1150 nm (62), respectively.…”

Thiosulfate Dehydrogenase (TsdA) from Allochromatium vinosum

“…It includes tyrosine in cytochrome f (16), the side chain of lysine in cytochrome c nitrite reductase (17) and its physiological partner NrfH (18), asparagine (19) in sphaeroides heme protein (19), and also cysteine. The latter was first discovered in the SoxXA protein of Rhodovulum sulfidophilum (20). In the catalytic subunit SoxA, the active site heme-ligating cysteine is modified by a further sulfur atom (i.e.…”