Extended heme promiscuity in the cyanobacterial cytochrome c oxidase: Characterization of native complexes containing hemes A, O, and D, respectively

The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocy stis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-so urces, respectively. In addition, Anabaena and Nostoc were grown under dini trogen-fixing conditions in a medium free of combined nitrogen. Membranes w ere isolated from late-logarithmic cells (culture density corresponding to approximately 3 mu l packed cells per milliliter); cytoplasmic and thylakoi d membranes were separated and purified according to established procedures . Acid-labile hemes were extracted from the membranes and subjected to reve rsed-phase highperformance liquid chromatography. Separated hemes were anal yzed spectroscopically and identified by comparison with authentic standard s. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally dis cernible heme D in the membranes and extracts therefrom. However, spectrosc opic and kinetic investigation of the membrane-bound heme D rather disprove d any reaction with oxygen or carbon monoxide. Kinetic measurements perform ed with the membrane-bound respiratory oxidase gave evidence for only two k inetically competent terminal oxidases, a(3) and o(3), both apparently asso ciated with a single type of apoprotein, viz. subunit I of the known cyanob acterial aa(3)-type cytochrome c oxidase. The heme D, on the other hand, se ems to form a spectrally distinguished, yet kinetically ill-defined hemopro tein complex which does not qualify as a fully functional d-type terminal o xidase on our (wild-type) cyanobacteria even after growth under semianaerob ic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under d initrogen-fixing conditions did not change this situation. Thus, we are lef t with (wild-type) cyanobacteria forming an unbranched respiratory chain wi th only a single type of terminal oxidase protein, viz. the known aa(3)-typ e cytochrome c oxidase. This oxidase, however, may incorporate different pr osthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox env ironment. In particular, however, conditions for expression of the two quin ol oxidases potentially and additionally coded for by the genome of, e.g., Synechocystis sp. PCC6803 (see http://www. kazusa.or.jp/cyano), have not ye t been found. (C) 1999 Academic Press.