Towards Understanding the Trafficking and Function of Iron and Titanium Ions in Organisms

Abstract

It is estimated that up to one third of all proteins are metalloproteins. These proteins have evolved to use the metals that are, or at least were at the time of their initial evolution, the most accessible. Some active centers of metalloenzymes resemble the structures of minerals presumed to be present in precipitates from hydrothermal solutions in the ocean billions of years ago. The metals in these proteins serve myriad purposes from structure to transport to catalysis. For these purposes organisms must find a way to incorporate, transport and possibly store the metal ions from the environment. Iron, among other metals, is used for all the before mentioned purposes but in oxic aqueous conditions is hydrolysis prone. Depending on its oxidation state iron is either insoluble or reacts to form reactive oxygen species and is dangerous to organisms. Organisms have thus evolved complex mechanisms to overcome the challenges of trafficking hydrolysis prone metals. This dissertation will focus on the study of the trafficking of hydrolysis prone iron and titanium by organisms, from metal selection to their use and storage. An examination of why metals are chosen, sequestration and transport of these metals, and use of the metals is presented. This research, as a whole, explores the cellular life cycle of hydrolysis prone metals. It is thought that the first uses of metals before their incorporation by organisms were at mineral surfaces. To this end it would be useful for the organism to be able to attach to the mineral surface. Rhodococcus ruber GIN-1 was isolated for its ability to selectively bind to TiO2 over other metal oxides. Biologically it could be advantageous to selectively bind to one mineral surface over another. The isolation and identification of these proteins are examined within. Rhodococcus ruber GIN-1 has also been found to produce a novel siderophore. The siderophore is not yet completely identified but falls into the class of catecholates. Once organisms begin to incorporate and use metals in proteins it would be useful to sequester and concentrate necessary metal ions that exist in low concentration in their environment. There are multiple organisms that are known to sequester high levels of titanium. One relatively unexplored family is that of Sabellidae or the feather duster worm. Organisms like this have been proposed as sentinel organisms to detect metal pollution in waters. In a model Sabellidae organism we have detected elevated levels of titanium, among other metals. After metal sequestration from the environment, intraorganism transport of the ions to where they are necessary becomes important. Higher organisms use the transferrin family of proteins to traffic iron. While the transferrin cycle has been studied in depth, the reduction mechanism has not been elucidated in detail. We use a monolobal transferrin, nicatransferrin, from the model organism Ciona intestinalis to explore this iron reduction mechanism of the transferrin cycle and find that nicatransferrin can reduce iron with no external reductant. This reduction occurs on the timescale expected for the transferrin cycle and occurs without an iron (II) chelator. The source of the reducing equivalent is unknown but nicatransferrin was measured to have reduced up to 2.5 equivalents of iron. Once transported to cells the metal ions can be put to use and incorporated into proteins or other structures. We examine the possible intentional use of titanium as a pigment in Eudistoma purpuropuntatum. The most abundant titanium sequesterer known is Eudistoma ritteri, who concentrates titanium up to 1500 ppm (dry weight). Eudistoma purpuropunctatum, a close relative of Eudistoma ritteri, displays an interesting purple color due to small granules in its tunic. We investigate the source of the purple color in these granules and the ability of the organism to sequester titanium, finding that it has titanium concentrations on par with Eudistoma ritteri. The metal ions that are not put to immediate use can be stored. Some metals exist in labile pools but due to iron’s reactivity it is necessary to store it where it cannot cause cellular damage. The iron storage protein ferritin is a cage-like polymer made up of 24 ferritin monomers. The monomers exist as either H-chain or L-chain and the 24-mer can be comprised of just one type of these monomers or a mixture thereof. The covalent dimerization of the human L-chain 24-mer has been observed and the cause of this dimerization explored. We do not find direct evidence of the covalent linkage but do identify regions of the protein most likely to participate in the dimerization.