Microsoft Word - 99999528-file00.docx

In contrast to other terrestrial arthropods where gaseous O2 that fuels aerobic metabolism diffuses to the tissues in tracheal tubes, and most other metazoans where O2 is transported to tissues by circulating respiratory proteins, the myriapods (millipedes and centipedes) strikingly have tracheal systems as well as circulating hemocyanin (Hc). In order to elucidate the evolutionary origin and biological significance of millipede Hc we report the molecular structure (subunit composition and amino acid sequence) of multimeric (36-mer) Hc from the forest-floor dwelling giant African millipede Archispirostreptus gigas and its allosteric oxygen binding properties under various physico-chemical conditions. A. gigas Hc consists of only a single subunit type with differential glycosylation. Phylogenic analysis reveals that millipede Hc is a sister group to centipede HcA, which supports an early divergence of distinct Hc subunits in myriapods and an ancient origin of multimeric Hcs. A. gigas Hc binds O2 with a high affinity and shows a strong normal Bohr effect. O2 binding is moreover modulated by Ca 2+ ions, which increase the O2 affinity of the Hc in the T (tense; deoxygenated) as well as the R (relaxed; oxygenated) states, and by (L)-lactate, which modulates Hc-O2 affinity by changing the allosteric equilibrium constant, L. Cooperativity in O2-binding at half O2-saturation (n50) is pH-dependent and maximal at pH ~7.4 and the number of interacting O2 binding sites (q) is markedly increased by binding Ca. The data is discussed in the light of role of mutually supplementary roles of Hc and the tracheal system for tissue O2 supply.