Reversible temperature-dependent high- to low-spin transition in the heme Fe–Cu binuclear center of cytochrome ba3 oxidase

A reversible temperature-dependent high- to low-spin transition with T1/2 = −60 °C has been observed in the resonance Raman spectra of the equilibrium reduced and photoreduced heme a3 of the thermophilic ba3 heme–copper oxidoreductase.

A Novel Heme a Insertion Factor Gene Cotranscribes with the Thermus thermophilus Cytochrome ba3 Oxidase Locus

ABSTRACT Studying the biogenesis of the Thermus thermophilus cytochrome ba 3 oxidase, we analyze heme a cofactor insertion into this membrane protein complex. Only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical Surf1 homologue, required for heme a insertion, is available from genome sequence data. Here, we characterize the product of an open reading frame, cbaX, in the operon encoding subunits of the ba 3-type cytochrome c oxidase. CbaX shares no sequence identity with any known oxidase biogenesis factor, and CbaX homologues are found only in the Thermaceae group. In a series of cbaX deletion and complementation experiments, we demonstrate that the resulting ba 3 oxidase complexes, affinity purified via an internally inserted His tag located in subunit I, are severely affected in their enzymatic activities and heme compositions in both the low- and high-spin sites. Thus, CbaX displays typical features of a generic Surf1 factor essential for binding and positioning the heme a moiety for correct assembly into the protein scaffold of oxidase subunit I.