Crystal structures of two hemoglobin components from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera)

pH-dependent structural changes in haemoglobin component V from the midge larvaPropsilocerus akamusi(Orthocladiinae, Diptera)

“…The crystal structure of P. akamusi Hb V at each pH was solved at 3.5 Å resolution by the molecular-replacement (MR) method with CNX (MSI Inc., California, USA). The search model used was the previously determined structure of Hb V (PDB entry 1x3k; Kuwada et al, 2007). To generate the search model, the residues of the haem pocket (18 amino-acid residues) were replaced with Ala and the haem group and all solvent atoms were removed.…”

“…By convention, individual residues were designated by the homologous helical position in sperm whale Mb (Knapp et al, 2005). Thus, we designated Ile66 as the E7 residue, which corresponds to HisE7 in Mb, and His98 as the F8 residue, although Ile66 and His98 are the sixth residue and the 15th residue in the E and F helices of Hb V, respectively (Kuwada et al, 2007). The bond angles and distances between the atoms were measured using Swiss-PdbViewer (Guex & Peitsch, 1997) and CONTACT (Collaborative Computational Project, Number 4, 1994).…”

“…Components V (Hb V) and VII (Hb VII) are representatives of the L-type and the N-type, respectively. The structural and functional features of these components have been examined in previous studies (Fukuda et al, 1993;Akiyama et al, 1994;Koshikawa et al, 1998;Kamimura et al, 2003;Yamamoto et al, 2003;Kuwada et al, 2007). One of the most remarkable differences between the two components is the replacement of HisE7 by a nonpolar Ile in Hb V. Kamimura et al (2003) demonstrated that despite this replacement Hb V exhibits oxygen affinity and a Bohr effect similar to those observed in mammalian Mbs.…”

“…They further predicted that PheB10 stabilizes the bound O 2 in Hb V. However, paramagnetic proton nuclear magnetic resonance (H-NMR) studies on methaemoglobin (met-Hb) V indicated the characteristic OH À and Cl À affinities to be pH-dependent and suggested that the side chain of ArgE10 acts as a proton donor for these ligands in the protein (Koshikawa et al, 1998;Yamamoto et al, 2003). In order to understand the structural features of the Hb components in insects, we have previously determined the crystal structures of Hb V and Hb VII from P. akamusi (Kuwada et al, 2007). Structural comparison of these Hb components indicated that Hb V has an additional helix at the C-terminal region, although Hb VII has a normal globin fold composed of eight helices, and the haem geometry in ligated Hb V differs from that in typical globin proteins.…”

“…Changes in the pH have a significant effect on the functional properties of Hb V, such as the Bohr effect and the anionic ligand affinity (Koshikawa et al, 1998;Kamimura et al, 2003;Yamamoto et al, 2003). The pH-dependence of its functional properties indicates that Hb V exhibits pH-dependent structural changes; therefore, crystallographic analysis of the protein at a given pH alone would not provide sufficient structural information (Kuwada et al, 2007). In fact, vertebrate Hbs have been reported to show significant structural changes with varying pH, with a consequent contribution to ligand affinity (Robinson et al, 2003;Yokoyama et al, 2004).…”