SOLUTION STRUCTURE OF HORSE HEART FERROCYTOCHROME-C DETERMINED BY HIGH-RESOLUTION NMR AND RESTRAINED SIMULATED ANNEALING

A model for the solution structure of horse heart ferrocytochrome c ha s been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty- four highly refined structures were obtained using a total of 1940 dis tance constraints based on the observed magnitude of nuclear Overhause r effects and 85 torsional angle restraints based on the magnitude of determined J-coupling constants. The all-residue root mean square devi ation about the average structure is 0.47 +/- 0.09 Angstrom for the ba ckbone N, C alpha, and C' atoms and 0.91 +/- 0.07 Angstrom for all hea vy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal st ructures of homologous c-type cytochromes. However, a detailed compari son between the model for the solution structure and the available mod el for the crystal structure of tuna ferrocytochrome c indicates signi ficant differences in a number of secondary and tertiary structural fe atures. For example, two of the three main helices display 3(10) to al pha-helical transitions resulting in bifurcation of main-chain hydroge n bond acceptor carbonyls. The N- and C-terminal helices are tightly p acked and display several interhelical interactions not seen in previo usly reported models. The geometry of heme ligation is well-defined an d completely consistent with the crystal structures of homologous cyto chromes c as are the locations of four of six structural water molecul es. Though the total solvent-accessible surface area of the protoporph yrin ring is similar to that seen in crystal studies of tuna ferrocyto chrome c, the distribution is somewhat different. This is mainly due t o a difference in packing of residues Phe-82 and Ile-81 such that Ile- 81 crosses the edge of the heme in the solution structure. These and o ther observations help to explain a range of physical and biological d ata spanning the redox properties, folding, molecular recognition, and stability of the protein.