J. Martin Bollinger, Jr.
Proteins that bind complex metal ion clusters play essential roles in such crucial processes as nitrogen fixation, photosynthesis, oxidative phosphorylation and ribonucleotide reduction. For these proteins, assembly and/or insertion of their metal ion cofactors is an essential post-translational processing event. In addition, it has become apparent in the last several years that assembly of metal clusters into proteins and the opposing process of disassembly occur dynamically in several toxicological, pharmacological, and normal physiological contexts. The convergence of chemical issues of metallocofactor assembly/disassembly and their biological implications is the focus of my research program. In general terms, my objectives are to understand 1) the molecular logic of metallocofactor assembly, disassembly and net stability and 2) the important biological effects, such as regulation of transcription and enzyme activity, that these processes mediate.
Figure 1: Schematic representations of some structurally characterized FeS Clusters, ranging from the simple Fe2S2 unit (left) to the complex P-cluster pair of dinitrogen reductase (right, adapted from Chan, et al., 1993, Science 260: 792-794).
Iron-sulfur (FeS) clusters are structurally diverse metallocofactors (Figure 1) that have been extensively studied for their ubiquitous roles in biological redox processes. It has recently become clear that assembly and disassembly of FeS clusters go on dynamically in several proteins in both regulatory and toxicological contexts. For example, oxidative disassembly of FeS clusters in certain essential enzymes is thought to be a primary mechanism of oxidant toxicity, including that induced in cancer cells by activated macrophages. Conversely, cluster assembly and disassembly appear to comprise a switching mechanism in several regulatory proteins, including iron regulatory factor, the post-transcriptional regulator of intracellular iron levels in mammals; FNR, the transcription factor of E. coli that senses O2 levels and appropriately regulates genes of primary metabolism; and SoxR, the transcription factor of E. coli that senses oxidative stress and initiates a global adaptive response. Recent studies on the post-translational processing of the enzyme dinitrogenase reductase (also known as nitrogenase Fe protein) from nitrogen-fixing bacteria have established the existence of a complex enzyme machinery to assemble its Fe4S4 cluster, and elements of this machinery are now known to be present in non-nitrogen-fixing species from bacteria to man. The simultaneous recognition 1) of dynamic FeS cluster assembly/disassembly as an important phenomenon and 2) of a general protein machinery dedicated to cluster assembly has opened a new area of investigation into mechanisms and physiological consequences of FeS cluster assembly and disassembly. Using nitrogenase Fe protein, SoxR, and FNR as model systems, my laboratory is seeking 1) to identify assembly/disassembly factors for FeS clusters (where they are not known), 2) to elucidate the chemical mechanisms of cluster assembly and/or disassembly, and 3) to use this mechanistic insight to understand structural determinants of the "engineered cluster instability" that appears to be the basis for transcriptional regulation by FNR and SoxR.
Figure 2. Schematic representation of the tyrosyl radical-diiron (III) cofactor in the R2 subunit of E. coli ribonucleotide reductase. (Adapted from Nordlund, et al. 1990, Nature 345: 593-598.
The objective of my second area of research is to understand the assembly of the tyrosyl radical-diiron(III) cofactor (Figure 2) of ribonucleotide reductase (RNR), the enzyme which provides all cells with the deoxynucleotide precursors for DNA synthesis. The RNR cofactor assembles spontaneously into the enzyme's R2 subunit when the apo form of the protein (lacking both iron and radical) is incubated with Fe(II) and O2. This assembly reaction has great medical relevance, as it opposes the activities of several anticancer and antiviral drugs (e.g. hydroxyurea, thiosemicarbazones), which reductively disassemble the cofactor and thereby inhibit DNA synthesis. Our previous investigation of the assembly reaction in E. coli RNR (see references below), which were the first to characterize the assembly mechanism of a complex metallocofactor, revealed as yet unexplored complexity in Fe(II) binding and electron gating by the protein. These are arguably the crucial issues relative to drug design (in view of the known drugs which reductively disassemble the cofactor). My laboratory is currently addressing these issues for cofactor assembly in E. coli RNR, and extending our previous studies to include the RNRs from more medically relevant mammalian and viral sources. Comparison of the assembly reactions in these three RNRs may shed light on known physical and chemical differences among their cofactors, and may suggest strategies for engineering selectivity in new cofactor-targeting drugs.