Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes

The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII–CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.

Folate-conjugated Helix lucorum hemocyanin – preparation, stability, and cytotoxicity

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance–Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 μg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 μg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.

Structure, function, and evolution of respiratory proteins

Respiratory proteins are complexes of proteins and metal ions. In haemoglobin the metal is iron, in haemocyanin—the most common invertebrate respiratory protein—it is copper. Globins such as haemoglobin and myoglobin and related molecular complexes have probably been around as long as life itself, whereas others such as the most common respiratory protein of molluscs and arthropods, haemocyanin, appear to be younger and are not chemically related to globins. Nevertheless, astounding functional similarities between haemoglobin and haemocyanin are seen. The present chapter takes a look at the molecular mechanisms behind their function, their fundamental integration in the respiratory process, and also traces the evolution of these respiratory proteins.

Jumping on the Edge—First Evidence for a 2 × 6-meric Hemocyanin in Springtails

Hemocyanins are respiratory dioxygen carrier proteins found in many arthropods including ancient terrestrial species such as spiders and scorpions as well as marine horseshoe crabs. As hemocyanins are highly conserved in this lineage, it is possible to observe an evolutionary descent through its subunits and their overall structure. Unfortunately, little is known about the structure and function of hexapod hemocyanins. Using recent springtail taxa (Collembola) as models for basal hexapods, and the help of electron microscopy, light scattering, SDS PAGE, and Western blot, we could demonstrate for the first time the presence of 2 × 6-meric hemocyanins in the hemolymph of hexapods. The quaternary structure is composed of at least two different subunits and looks nearly identical to the hemocyanin found in decapod crustaceans. In addition, homology modeling and western blotting suggest a close structural relationship between collembolan and crustacean hemocyanin. Such a respiratory protein was possibly helpful in the early terrestrialization process of ancient Collembola. In addition, physiological adaptations to hypoxic or temporarily anoxic conditions could be a possible explanation for the presence of this respiratory protein. Nevertheless, it has to be concluded that the primary benefit of hemocyanin for springtails remains unclear.