Honami Yamashita

Alternative structural state of transferrin. The crystallographic analysis of iron-loaded but domain-opened ovotransferrin N-lobe.

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO3 2−. Upon Fe3+uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121.

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe3+ release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe3+ release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe3+ with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe3+ release model in which the holo form is converted depending on the anion concentration into a “mixed ligand” intermediate that releases Fe3+. The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 Å resolution demonstrated the existence of a single bound SO 4 2 − in the interdomain cleft, which interacts directly with Thr461-OG1, Tyr431-OH, and His592-NE2 and indirectly with Tyr524-OH. The latter three groups are Fe3+-coordinating ligands, strongly suggesting the facilitated Fe3+ release upon the anion occupation at this site. The SO 4 2 − binding structure supported the single-pathway kinetic model.

Involvement of Ovotransferrin in the Thermally Induced Gelation of Egg White at around 65°C.

Egg white forms a soft opaque gel at around 65°C. An analysis of the turbidity appearance in the presence or absence of iron and polyacrylamide gel electrophoresis of the precipitated proteins after thermal treatment revealed ovotransferrin to be the major component involved in thermal gelation at around 65°C. The storage modulus of the thermally induced gel of purified ovotransferrin reinforced this conclusion.

Post-harvest changes in the respiratory activity of and ethanol accumulation in fresh rice grains

Fresh rice grains stored under anaerobic conditions at 4°C showed a strong activity of anaerobic respiration at 30°C. When stored at 30°C, the rates of both oxygen consumption and carbon dioxide evolution declined rapidly. The ethanol content in paddy at the post-harvest stage was increased at 4°C, whereas no significant accumulation of ethanol was observed at 30°C. The accumulated ethanol in paddy was depleted as the storage temperature was raised from 4 to 30°C. In contrast, a temperature-dependent accumulation was observed with a lowering from 30 to 4°C. On the other hand, ethanol content in brown rice changed little with storage temperature. On the basis of these results, it is assumed that ethanol is more easily accumulated in the rice grains against diffusion to the atmosphere at the lower temperature.