Metallation and mismetallation of iron and manganese proteins in vitro and in vivo: the class I ribonucleotide reductases as a case study

How cells ensure correct metallation of a given protein and whether a degree of promiscuity in metal binding has evolved are largely unanswered questions. In a classic case, iron- and manganese-dependent superoxide dismutases (SODs) catalyze the disproportionation of superoxide using highly similar protein scaffolds and nearly identical active sites. However, most of these enzymes are active with only one metal, although both metals can bind in vitro and in vivo. Iron(II) and manganese(II) bind weakly to most proteins and possess similar coordination preferences. Their distinct redox properties suggest that they are unlikely to be interchangeable in biological systems except when they function in Lewis acid catalytic roles, yet recent work suggests this is not always the case. This review summarizes the diversity of ways in which iron and manganese are substituted in similar or identical protein frameworks. As models, we discuss (1) enzymes, such as epimerases, thought to use Fe[superscript II] as a Lewis acid under normal growth conditions but which switch to Mn[superscript II] under oxidative stress; (2) extradiol dioxygenases, which have been found to use both Fe[superscript II] and Mn[superscript II], the redox role of which in catalysis remains to be elucidated; (3) SODs, which use redox chemistry and are generally metal-specific; and (4) the class I ribonucleotide reductases (RNRs), which have evolved unique biosynthetic pathways to control metallation. The primary focus is the class Ib RNRs, which can catalyze formation of a stable radical on a tyrosine residue in their β2 subunits using either a di-iron or a recently characterized dimanganese cofactor. The physiological roles of enzymes that can switch between iron and manganese cofactors are discussed, as are insights obtained from the studies of many groups regarding iron and manganese homeostasis and the divergent and convergent strategies organisms use for control of protein metallation. We propose that, in many of the systems discussed, “discrimination” between metals is not performed by the protein itself, but it is instead determined by the environment in which the protein is expressed.National Institutes of Health (U.S.) (Grant GM81393

Protein-Based Platform for Purification, Chelation, and Study of Medical Radiometals: Yttrium and Actinium

Actinium-based therapies could revolutionize cancer medicine but remain tantalizing due to the difficulties in studying Ac chemistry. Current efforts focus on small synthetic chelators, limiting radioisotope complexation and purification efficiencies. Here we demonstrate how a recently discovered protein, lanmodulin, can be utilized to efficiently bind, recover, and purify medically-relevant radiometals, actinium(III) and yttrium(III), and probe their chemistry. The stoichiometry, solution behavior, and formation constant of the 228Ac-lanmodulin complex (Ac3LanM, Kd, 865 femtomolar) and its 90Y/natY/natLa analogues were experimentally determined, representing both the first actinium-protein and most stable actinide(III)-protein species to be characterized. Lanmodulin’s unparalleled properties enable the facile purification-recovery of radiometals, even in the presence of >10+10 equivalents of competing ions and at ultra-trace levels: down to 2 femtograms 90Y and 40 attograms 228Ac. The lanmodulin-based approach charts a new course to study elusive isotopes and develop versatile chelating platforms for medical radiometals, both for high-value separations and potentially in vivo applications

Synthetic fluorescent probes for studying copper in biological systems

The potent redox activity of copper is required for sustaining life. Mismanagement of its cellular pools, however, can result in oxidative stress and damage connected to aging, neurodegenerative diseases, and metabolic disorders. Therefore, copper homeostasis is tightly regulated by cells and tissues. Whereas copper and other transition metal ions are commonly thought of as static cofactors buried within protein active sites, emerging data points to the presence of additional loosely bound, labile pools that can participate in dynamic signalling pathways. Against this backdrop, we review advances in sensing labile copper pools and understanding their functions using synthetic fluorescent indicators. Following brief introductions to cellular copper homeostasis and considerations in sensor design, we survey available fluorescent copper probes and evaluate their properties in the context of their utility as effective biological screening tools. We emphasize the need for combined chemical and biological evaluation of these reagents, as well as the value of complementing probe data with other techniques for characterizing the different pools of metal ions in biological systems. This holistic approach will maximize the exciting opportunities for these and related chemical technologies in the study and discovery of novel biology of metals