|dc.description.abstract||Iron-sulfur-cluster-containing proteins are present in all living organisms. They are involved in a wide array of biological processes such as electron transfer, substrate binding and activation, regulation of enzyme activity and gene expression, sensing of reactive species, radical generation and sulfur donation. The work in this dissertation is focused on the spectroscopic characterization of two proteins that contain [4Fe-4S] clusters in their active site, (E)-4-hydroxy-
3-methylbut-2-enyl diphosphate reductase (IspH) and subunit A of heterodisulfide reductase (HdrA).
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase is the terminal enzyme in the biosynthesis of isoprenoids via the 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway. IspH catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into two five carbon products, isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP). The DOXP pathway is responsible for the biosynthesis of isoprenoids in many pathogenic eubacteria, plastid containing eukaryotes, and parasitic protozoans. Humans use a different pathway, making the DOXP route a promising target for the development of herbicides and antimicrobials. To develop potential inhibitors for the DOXP pathway the catalytic mechanism of IspH has been investigated. Colorimetric assays, site-directed mutagenesis, and spectroscopic methods (EPR, ENDOR and Mӧssbauer) have been employed in this work to study the role of the [4Fe-4S] cluster and several conserved amino acid residues in the active site of the enzyme. EPR-monitored rapid-freeze-quench experiments with wild type IspH under turnover conditions in the presence of a reductant and substrate showed the formation of two paramagnetic species. The first signal denoted FeS-I, was further characterized with 57Fe-ENDOR and Mӧssbauer spectroscopies. The electronic and magnetic properties of this intermediate are similar to a HiPIP-like [4Fe-4S]3+ species that was detected in ferredoxin:thioredoxin reductase, indicating the direct binding of the substrate to a cluster that has a formal 3+ oxidation state. This species is also similar to an EPR active intermediate that was trapped in IspG, another DOXP pathway enzyme, and heterodisulfide reductase. The second
species, FeS-II, represents a cluster with product bound because a similar signal was observed when dithionite-reduced enzyme was incubated with IPP or DMAPP.
In EPR-monitored rapid-freeze-quench experiments with mutant IspH (H124F, E126Q and T167C) similar and additional paramagnetic intermediates were observed. When reduced H124F and T167C IspH were incubated with HMBPP, two EPR active intermediates are detected that are similar to the species in the WT enzyme. This is in line with the fact that both of these mutants were active, albeit with lower catalytic activity. The E126Q mutant on the other
hand, has no activity and a paramagnetic species (designated FeS-III) that is different from the EPR active species in the WT enzyme can be detected. When dithionite-reduced E126Q IspH was incubated with IPP or DMAPP, an identical signal was observed.
The FeS-III species detected in the E126Q mutant is considered a reaction intermediate by several groups and several hypothetical mechanisms are based on the electronic and magnetic properties of this species. Our freeze-quench data, however, suggests that this species appears much later in the reaction than the FeS-I species, which makes the catalytic relevance of this species questionable. The kinetic and spectroscopic analyses are in line with an important role in catalysis for some of the mutated amino acids. H124 and T167 are proposed to participate in the correct orientation of the substrate, while the E126 residue is involved in the dehydration of the HMBPP. Although the kinetic data obtained here does not argue against the current reaction mechanism, there is the real concern that some aspects are based on the properties of the FeS-III species detected in the E126Q mutant and not on the properties of the true reaction intermediate, FeS-I. This species needs to be further investigated, particularly using ENDOR studies of IspH incubated with 13C-labeled HMBPP.
Heterodisulfide reductase, also referred as Hdr, is an iron-sulfur flavoprotein that catalyzes the reversible cleavage of CoM-S-S-CoB. Heterodisulfide reductase is involved in the flavin-based electron bifurcation mode of energy conservation in methanogenic archaea. Since the Hdr enzyme contains up to 7 iron-sulfur clusters, it was investigated if it was possible to just study the A subunit. This subunit harbors FAD, which is the supposed site of the bifurcation.
HdrA, from M. marburgensis was overexpressed in M. maripaludis and was successfully purified. The enzyme contained substoichiometric amounts of [4Fe-4S] clusters and in vitro cluster reconstitution successfully increased the cluster content. FAD was absent and attempts to reconstitute it were not successful. EPR-based redox titration of HdrA failed to show a particular paramagnetic species that was detected in the redox titration of Hdr and the whole
Hydrogenase/Hdr complex. This cluster is proposed to be the direct electron acceptor for the high-potential electron from the FAD. The absence of the EPR signature indicates that the HdrA subunit cannot be used in these studies.||en_US