ADAPTATION TO EXTREME ENVIRONMENTS: INSIGHTS FROM HALOPHILIC FERREDOXIN AND ACIDOPHILIC RUSTICYANIN. Menachem Shoham and Dong Zhao, Case Western Reserve University School of Medicine, Department of Biochemistry, Cleveland, Ohio 44106-4935.

What causes a protein to withstand extremes in pH or to remain soluble in saturated salt? Some insights can be gained by comparing the crystal structures of extremophilic proteins with those of other members of the same family.

The crystal structure of a halophilic 2Fe-2S ferredoxin from Haloarcula marismortui suggests two mechanisms for keeping this protein soluble and active at the saturated salt solution prevailing in the cytosol of this archaebacterium: 1. halophilic substitution of polar uncharged surface residues by aspartic and glutamic acid; 2. halophilic addition of an extra domain consisting of two amphipathic helices and intervening loops. This domain is inserted in between the two antiparallel [[beta]] strands 1 and 2 instead of the [[beta]] hairpin found at this position in plant-type 2Fe-2S ferredoxins. The surface of this domain is entirely made up of 15 carboxylates. Glutamic and aspartic acid are known to be the best water-binding residues amongst the 20 naturally occurring amino acids. The postulated function of this domain is to increase the water-binding capacity of the protein, thus enabling the protein to compete effectively with the multitude of inorganic cations for solvation. Turning the protein into a large polyanion of net charge -28 prevents self aggregation and keeps the protein in solution.

Rusticyanin from Thiobacillus ferrooxidans is a type I blue-copper protein stable and active in sulfuric acid at pH values as low as 0.2. The crystal structure of rusticyanin reveals several buried chargeable residues. The two histidine copper ligands are shielded from solvent by interactions with nearby hydrophobic residues. Some aspartic acid residues, notably Asp 88, located near the copper-binding site, are buried inside the protein. These carboxyls are postulated to be in uncharged form and will have pK_a values quite different from the values in free solution. The net effect on the protein is to shift the pH profile of stability towards the more acid range.