Importance of nitrate in the crystal growth of cytochrome c from four biological species judged by morphodrom analysis

Crystallization of cytochrome c from horse, bovine, tuna and yeast (Iso-1) sources was attempted under similar conditions using ammonium sulfate and s odium nitrate as crystallization agents. With the exception of yeast cytoch rome c, we were successful in obtaining crystals, however, in different for ms. The requirement for similar amounts of the two salts suggested the impo rtance of sodium nitrate for crystallization. That is, each salt plays a di fferent role in the crystallization process: ammonium sulfate providing the ionic strength for the salting-out, sodium nitrate the specific intermolec ular contacts. X-ray crystallographic analysis of the trigonal crystals of the horse protein revealed the existence of 2.5 nitrate ions per cytochrome c molecule in the crystal lattice. Morphodroms were made versus the concen trations of both salts. Their comparison clearly showed similarities and di fferences of the crystal habits among horse, bovine and tuna. The differenc es in the morphodroms correlated with the evolutionary divergence of the sp ecies. It was suggested that Asn103 played an important role in the crystal packing by interacting with one of the nitrate ions. This explains why, in the history of structure determination of this protein, only the crystals that were formed in the presence of two kinds of salts diffracted over 2.0 Angstrom resolution. (C) 2001 Published by Elsevier Science B.V.