MULTIPLE WAVELENGTH ANOMALOUS DIFFRACTION (MAD) CRYSTAL-STRUCTURE OF RUSTICYANIN - A HIGHLY OXIDIZING CUPREDOXIN WITH EXTREME ACID STABILITY

The X-ray crystal structure of the oxidized form of the extremely stab le and highly oxidizing cupredoxin rusticyanin from Thiobacillus ferro oxidans has been determined by the method of multiwavelength anomalous diffraction (MAD) and refined to 1.9 Angstrom resolution. Like other cupredoxins, rusticyanin is a copper-containing metalloprotein, which is composed of a core beta-sandwich fold. In rusticyanin the beta-sand wich is composed of a six- and a seven-stranded beta-sheet. Also like other cupredoxins, the copper ion is coordinated by a cluster of four conserved residues (His85, Cys138, His143, Met148) arranged in a disto rted tetrahedron. Rusticyanin has a redox potential of 680 mV, roughly twice that of any other cupredoxin, and it is optimally active at pH values less than or equal to 2. By comparison with other cupredoxins, the three-dimensional structure of rusticyanin reveals several possibl e sources of the chemical differences, including more ordered secondar y structure and more intersheet connectivity than other cupredoxins. T he acid stability and redox potential of rusticyanin may also be enhan ced over other cupredoxins by a more extensive internal hydrogen bondi ng network and by more extensive hydrophobic interactions surrounding the copper binding site. Finally, reduction in the number of charged r esidues surrounding the active site may also make a major contribution to acid stability. We propose that the resulting rigid copper binding site, which is constrained by the surrounding hydrophobic environment , structurally and electronically favours Cu(I). We propose that the t wo extreme chemical properties of rusticyanin are interrelated; the sa me unique structural features that enhance acid stability also lead to elevated redox potential. (C) 1996 Academic Press Limited