The structure of cytochrome b562 from E. coli and of flavocytochrome b2 [L-(+)- lactate dehydrogenase] from bakers yeast will be studied by x-ray diffraction methods. Both the oxidized and reduced forms of each protein will be examined. The major goal will be to understand the mechanism of electron transport at the molecular level. The structure of ferricytochrome b562 has been solved at 2.5A resolution by single isomorphous replacement with anomalous scattering and 2-fold electron density averaging. The ligands to the noncovalently bound heme irons are histidine and methionine. The protein consists of four nearly parallel Alpha-helicces and resembles cytochrome c', thus defining a new class of cytochromone fold. The phases have been extended to 2.0A and the protein will be refined at 2.0A and then at 1.5A resolution. The reduced and high pH forms of the protein will be crystallized and analyzed at atomic resolution. Flavocytochrome b2 contains both a flavin and a heme prosthetic group. It catalyzes the dehydrogenation of L-lactate, transferring electrons to cytochrome c in the mitochondria. It contains a trypsin resistant heme peptide core which resembles microsomal cytochrome b5 in sequence and spectrum. One goal of this research is to learn the relative positions of the heme and flavin groups in b2 and the structural basis for catalysis and subsequent electron transfer. A second goal is to learn the structural and evoluationary relationship between b2 and b5. Cytochromoe b2 crystallizes in a trigonal cell with one tetramer of 230,000 daltons per asymmetric unit. The isomorphous replacement method of structure investigation will be coupled with the molecular replacement method to solve this structure. The structure-function relationships and comparison with other proteins, as well as electron density fitting, will be carried out on the MMS-X molecular graphics system.
