This project aims to delineate structural features that are responsible for recognition and reactivity in proteins, using high- resolution x-ray structure analysis as a primary method. The structure determinations focus on a series of flavin- and metal-containing enzymes and employ genetic engineering, spectroscopic, and biochemical techniques, in combination with x- ray crystallography, to describe e.g. how proteins modulate redox potentials and utilize conformational equilibria to control reactivity. Studies of redox- and ligation-linked conformation changes will exploit site directed modification of known structures. In continuing analyses of flavodoxins, mutation and computation will assess the roles of electrostatic interactions and conformation changes in controlling redox potentials. The rate of intramolecular electron transfer in the iron- sulfur flavoprotein, phthalate dioxygenase reductase (PDR), may be regulated by pyridine nucleotide binding or conformation-dependent interactions among cofactors. The mechanisms will be investigated by analysis of mutants and of the truncated protein, PDR (-Fe/S). The nature of the conformation changes that accompany pyridine nucleotide or substrate binding will be determined in PDR, in thioredoxin reductase and in p-hydroxybenzoate hydroxylase. Mutants and metal-ligated species of Fe- and Mn-superoxide dismutases will be examined to understand the structural chemistry of the metals in these oxygen scavengers and to assess proposed mechanisms for the coupling of electron with proton uptake. The structure of the central B12-binding region of E. coli methionine synthase has been determined as part of this project. De novo structure analyses of the N- and C-terminal regions of this 136 kDa protein will ascertain how substrates are bound and how each of three different methyl transfer reactions occurs at cobalamin. Impairment of mammalian methionine synthase, for which the E. coli enzyme serves as a model, is responsible for many of the manifestations of B12 deficiency. Crystallographic analysis of phthalate dioxygenase, a 200,000 kD multimeric enzyme that utilizes Fe2+ in reductive activation of oxygen for insertion into aromatic substrates, will determine the geometry and interactions of the novel mononuclear Fe2+ and of a Rieske [2Fe-2S] center. Cytidylyl transfer and recognition, key events in activation of the intermediates in lipid biosynthesis, will be studied in a soluble model enzyme from B. subtilis. Attempts will be made to crystallize nitric oxide synthase and some of its subfragments and to crystallize other metalloproteins, including the Rieske [2Fe-2S] protein from T. thermophilus.
