An archaeal b-type cytochrome containing a nonfunctional carbonic anhydrase-like domain.

A new type of cytochrome b was isolated from the cytoplasmatic fraction of the archaeon Acidianus ambivalens, which is the first soluble cytochrome found in this member of the thermoacidophilic order of the Sulfolobales. The protein is a monomeric and monohemic cytochrome b with a molecular mass of 22 kDa. Visible spectroscopy of the as-purified protein shows a Soret peak at 405 nm and a broad band at 625 nm, indicating the presence of a high-spin ferric heme. Upon reduction, the Soret band shifts to 422 nm and a broad band at 560 nm develops, again characteristic of high-spin ferrous heme. The reduced form can bind carbon monoxide, with visible absorption bands arising at 411 and 566 nm. EPR spectroscopy of the oxidized protein shows a spectrum typical of a high-spin heme, with major g values at 6.56 and 5.85. The reduction potential of the heme cofactor was determined to be -16+/-10 mV, at pH 6.5. Analysis of the protein amino acid sequence shows that it consists of a novel arrangement of domains. The first domain, at the N-terminus, has a remarkable similarity towards beta class carbonic anhydrases, whereas the second region comprises a putative cytochrome domain. The latter presumably consists of a novel fold, as it bears no sequence similarities towards other known cytochromes, or towards known domains. Strikingly, the first module contains the C-X (n)-H-X(2)-C motif that accounts for the binding of the catalytic zinc in carbonic anhydrases, but lacks several other critical residues required for substrate binding and proper active site geometry. In agreement with this finding, the isolated cytochrome contains one bound zinc atom, but has no carbonic anhydrase activity. Inspection of the sequences available from the genomic sequencing project of the close relative archaeon Sulfolobus solfataricus shows the presence of an identical protein, suggesting its dissemination among the Sulfolobales. The role of zinc as a key element for the intrinsic thermal stability of these proteins is discussed.