Two proteins, rubrerythrin (Rr) and hemerythrin (Hr), are proposed for study. Both of these proteins contain non-heme diiron sites with the latter containing in addition a rubredoxin (Rd)-type FeS4 site. The former protein appears to be involved in O2 activation or scavenging, whereas the latter is known to bind O2 reversibly. Structural and functional characterizations of these iron sites and their interactions with the surrounding polypeptides is the overall focus of the proposed research. The study of these two proteins together is designed to foster a synergism in understanding the properties of their similar, yet distinct diiron sites. Rr was originally isolated from Desulfovibrio vulgaris, a strictly anaerobic, sulfate-reducing bacterium. Its biological function and the structures of its metal sites have not yet been established. However, the gene sequence for Rr, together with spectroscopic and biochemical results allow several hypotheses to be put forward. These hypotheses include the following: i) the diiron site of Rr reacts with peroxides to form a high-valent species capable of oxidizing certain electron donors, i.e., Rr can function as a peroxidase. ii) two conserved amino acid sequences in Rr furnish ligands to the diiron site. iii) the Rd-type site functions as an electron donor/acceptor for the diiron site in Rr. iv) the Rr gene is part of an operon, at least one of whose proteins functions as electron donor/acceptor for Rr. Experimental tests of these hypotheses include sequencing of Rr genes from at least two other species, sequencing and overexpression of regions flanking the D. vulgaris Rr gene, characterization of a reconstituted Rr which is enriched in diiron sites, and site-directed mutagenesis of Rr that has been cloned and overexpressed in E. coli. Peroxidase activities of the reconstituted and recombinant, mutated Hrs will show whether this activity requires diiron sites. Site-directed mutagenesis of recombinant Hr is proposed in order to probe in detail the requirements for reversible O2 binding. Histidine ligands will be replaced with carboxylates, and side-chains capable of hydrogen bonding to the iron ligands will be altered. These changes should have predictable effects on O2 affinity. Exposure of the O2 binding site to solvent by substitution of less bulky surrounding side chains could either raise or lower O2 affinity depending on the relative effects of solvent exposure on O2 dissociation vs association rates. These predictions will be tested by measuring the O2 affinities and O2 dissociation rates of the mutated Hrs. Attempts will also be made to change the diiron site of Hr into one that activates O2 by removal of one of the bridging carboxylates and replacement of some of the histidine ligands with carboxylates. Hrs from almost all species do not bind O2 in a cooperative manner, but Hr from the brachiopod Lingula reevii is an exception. The discovery that two non-identical subunits are present in approximately equal amounts in this Hr raises the possibility that the cooperativity is due to an alpha4beta4 subunit composition. This hypothesis will be tested by separately cloning the alpha and beta subunits and measuring the O2 binding curves of the two types of subunits. These studies are aimed at a clearer understanding of O2 binding and O2 activation in biological systems.