Microbes vary strategically in their metalation of mononuclear enzymes.

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.

The vulnerability of radical SAM enzymes to oxidants and soft metals.

Radical S-adenosylmethionine enzymes (RSEs) drive diverse biological processes by catalyzing chemically difficult reactions. Each of these enzymes uses a solvent-exposed [4Fe-4S] cluster to coordinate and cleave its SAM co-reactant. This cluster is destroyed during oxic handling, forcing investigators to work with these enzymes under anoxic conditions. Analogous substrate-binding [4Fe-4S] clusters in dehydratases are similarly sensitive to oxygen in vitro; they are also extremely vulnerable to reactive oxygen species (ROS) in vitro and in vivo. These observations suggested that ROS might similarly poison RSEs. This conjecture received apparent support by the observation that when E. coli experiences hydrogen peroxide stress, it induces a cluster-free isozyme of the RSE HemN. In the present study, surprisingly, the purified RSEs viperin and HemN proved quite resistant to peroxide and superoxide in vitro. Furthermore, pathways that require RSEs remained active inside E. coli cells that were acutely stressed by hydrogen peroxide and superoxide. Viperin, but not HemN, was gradually poisoned by molecular oxygen in vitro, forming an apparent [3Fe-4S]+ form that was readily reactivated. The modest rate of damage, and the known ability of cells to repair [3Fe-4S]+ clusters, suggest why these RSEs remain functional inside fully aerated organisms. In contrast, copper(I) damaged HemN and viperin in vitro as readily as it did fumarase, a known target of copper toxicity inside E. coli. Excess intracellular copper also impaired RSE-dependent biosynthetic processes. These data indicate that RSEs may be targets of copper stress but not of reactive oxygen species.

Evolutionary adaptations that enable enzymes to tolerate oxidative stress.

Biochemical mechanisms emerged and were integrated into the metabolic plan of cellular life long before molecular oxygen accumulated in the biosphere. When oxygen levels finaly rose, they threatened specific types of enzymes: those that use organic radicals as catalysts, and those that depend upon iron centers. Nature has found ways to ensure that such enzymes are still used by contemporary organisms. In some cases they are restricted to microbes that reside in anoxic habitats, but in others they manage to function inside aerobic cells. In the latter case, it is frequently true that the ancestral enzyme has been modified to fend off poisoning. In this review we survey a range of protein adaptations that permit radical-based and low-potential iron chemistry to succeed in oxic environments. In many cases, accessory domains shield the vulnerable radical or metal center from oxygen. In others, the structures of iron cofactors evolved to less oxidizable forms, or alternative metals replaced iron altogether. The overarching view is that some classes of biochemical mechanism are intrinsically incompatible with the presence of oxygen. The structural modification of target enzymes is an under-recognized response to this problem.