Mechanism of biosynthesis of the dimanganese-tyrosyl radical cofactor of class lb Ribonucleotide reductase

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Ribonucleotide reductases (RNRs) catalyze the reduction of nucleotides to deoxynucleotides in all organisms. The class Ia and lb RNRs comprise two subunits: a2 contains the site of nucleotide reduction, and p2 contains an essential stable tyrosyl radical (Y·), generated by oxidation of a dinuclear metal cluster. The diferric-Y (Fe" 2-Y·) cofactor of the class Ia RNRs self-assembles by reaction of Fe"2-NrdB with 02 and a reducing equivalent. Whether the class Ib RNRs utilize a diiron or dimanganese cofactor in vivo has been controversial. To determine the physiological metallocofactor of the Escherichia coli class lb RNR, we recombinantly express and purify a2 (NrdE) and p2 (NrdF) and show that NrdF self-assembles an active Fe 12- Y· cofactor using Fe" and 02. We also present the first purification of NrdI, a protein of unknown function conserved in class lb RNR systems. We show that NrdI is a flavodoxin-like protein with unusual redox properties. Although Mnr 2-NrdF does not react with 02, in the presence of reduced NrdI (Nrdlhq) and 02, it assembles an active dimanganese(III)-Y· (Mn 12- Y·) cofactor. Biochemical evidence indicates that Nrdlhq binds tightly to NrdF and reacts with 02 to provide an oxidant that channels to the metal site in NrdF to assemble the Mn"12-Ycofactor, a model supported by crystal structures of a Mn"2-NrdF*NrdI complex. NrdF purified from its endogenous levels in an iron-limited E. coli strain contains the Mn" 2 -Y· cofactor, establishing its physiological relevance. Rapid kinetics studies of Mn"'12 -Y· cofactor assembly in Bacillus subtilis NrdF support a mechanism in which NrdIhg rapidly reduces 02 to 02- and the 02'- channels to and reacts with Mn"2-NrdF to form a Mn" Mnv intermediate, which oxidizes tyrosine to Y·. Finally, we also demonstrate that E. coli NrdF, when incubated anaerobically with Mn" and Fe" and then exposed to H202 , forms an active Y·-containing metallocofactor that we suggest is Fe"Mn'l-Y·. These results raise the issues of how a single active site can generate a stable, active Ye using three different metal cofactors and oxidants in vitro, and therefore how metallation of NrdF with manganese is controlled in vivo.by Joseph Alfred Cotruvo, Jr..Ph.D

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

Design of pure heterodinuclear lanthanoid cryptate complexes

Heterolanthanide complexes are difficult to synthesize owing to the similar chemistry of the lanthanide ions. Consequently, very few purely heterolanthanide complexes have been synthesized. This is despite the fact that such complexes hold interesting optical and magnetic properties. To fine-tune these properties, it is important that one can choose complexes with any given combination of lanthanides. Herein we report a synthetic procedure which yields pure heterodinuclear lanthanide cryptates LnLn*LX(3) (X = NO(3)(−) or OTf(−)) based on the cryptand H(3)L = N[(CH(2))(2)N[double bond, length as m-dash]CH–R–CH[double bond, length as m-dash]N–(CH(2))(2)](3)N (R = m-C(6)H(2)OH-2-Me-5). In the synthesis the choice of counter ion and solvent proves crucial in controlling the Ln–Ln* composition. Choosing the optimal solvent and counter ion afford pure heterodinuclear complexes with any given combination of Gd(iii)–Lu(iii) including Y(iii). To demonstrate the versatility of the synthesis all dinuclear combinations of Y(iii), Gd(iii), Yb(iii) and Lu(iii) were synthesized resulting in 10 novel complexes of the form LnLn*L(OTf)(3) with LnLn* = YbGd 1, YbY 2, YbLu 3, YbYb 4, LuGd 5, LuY 6, LuLu 7, YGd 8, YY 9 and GdGd 10. Through the use of (1)H, (13)C NMR and mass spectrometry the heterodinuclear nature of YbGd, YbY, YbLu, LuGd, LuY and YGd was confirmed. Crystal structures of LnLn*L(NO(3))(3) reveal short Ln–Ln distances of ∼3.5 Å. Using SQUID magnetometry the exchange coupling between the lanthanide ions was found to be anti-ferromagnetic for GdGd and YbYb while ferromagnetic for YbGd

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