Extraordinary stability of hemocyanins from L. polyphemus and E. californicum studied using infrared spectroscopy from 294 to 20 K.

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. Here we give infrared spectroscopic evidence of a high stability towards exposure to sub-zero temperatures for hemocyanins from the arthropods Limulus polyphemus and Eurypelma californicum at different pH values. Small but distinct temperature induced changes of the secondary structure were observed, but a stable core of at least 40% α-helical structure is preserved as identified in the infrared spectra obtained between 294 and 20 K. The structural changes differ in detail somewhat for the two hemocyanins, with overall fewer changes observed in the case of E. californicum. Notably, in both cases the overall changes in the α-helical content are found to be fully reversible. The small changes in the secondary structure and reversibility upon cold treatment seem to be a particular property of the two hemocyanins, since it was not observed for myoglobin studied in the same way.

Raman and infrared spectroscopic evidence for the structural changes of the 2Fe2S cluster and its environment during the interaction of adrenodoxin and adrenodoxin reductase.

Many biological functions involve the formation of protein-protein complexes. In the present study, we investigated the interaction of two proteins involved in electron transfer, adrenodoxin (Adx) and adrenodoxin reductase (AdR) by using Raman and infrared spectroscopies. Different shifts and splittings of the FeSb/t stretching vibrational modes upon interaction of the two proteins can be reported pointing towards major structural changes in the [2Fe2S] cluster. These changes may be necessary for optimizing electron transfer. The assignment of the shifted modes to the [2Fe2S] cluster was confirmed by 54Fe labeling of the truncated Adx (4-108) as well as the investigation of mutants close to the interaction site and in the vicinity of the [2Fe2S] cluster. Electrochemically induced FTIR difference spectra revealed that the flavin cofactor in AdR also changes due to the interaction with [2Fe2S] cluster in the Adx/AdR electron transfer complex.