title: Inhibition of the 4Fe-4S Proteins IspG and IspH: an EPR, ENDOR and HYSCORE Investigation.
abstract: IspG and IspH are proteins that are involved in isoprenoid biosynthesis in most bacteria as well as in malaria parasites and are important drug targets. They contain cubane-type 4Fe-4S clusters that are involved in unusual 2H+/2e-reductions. Here, we report the results of electron paramagnetic resonance spectroscopic investigations of the binding of amino- and thiolo-HMBPP (HMBPP=E-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate) IspH substrate-analog inhibitors to both proteins, as well as the binding of HMBPP and an acetylene diphosphate inhibitor, to IspG. The results show that amino-HMBPP binds to reduced IspH by Fe-C π-bonding with the olefinic carbons interacting with the unique 4thFe in the 4Fe-4S cluster, quite different to the direct Fe-N ligation seen with the oxidized protein. No such π-complex is observed when amino-HMBPP binds to reduced IspG. No EPR signal is observed with IspH in the presence of dithionite and thiolo-HMBPP, suggesting that the 4Fe-4S cluster is not reduced, consistent with the presence of a 420 nm feature in the absorption spectrum (characteristic of an oxidized cluster). However, with IspG, the EPR spectrum in the presence of dithionite and thiolo-HMBPP is very similar to that seen with HMBPP. The binding of HMBPP to IspG was studied using hyperfine sublevel correlation spectroscopy with17O and13C labeled samples: the results rule out direct Fe-O bonding and indicate π-bonding. Finally, the binding to IspG of a potent acetylene diphosphate inhibitor was studied by using electron-nuclear double resonance spectroscopy with13C labeled ligands: the large hyperfine couplings indicate strong Fe-C π-bonding with the acetylenic group. These results illustrate a remarkable diversity in binding behavior for HMBPP-analog inhibitors, opening up new routes to inhibitor design of interest in the context of anti-bacterial and anti-malarial drug discovery, as well as "cubane-type" metallo-biochemistry, in general.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 607 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Francisco
authors.initials: F
authors.lastname: Guerra
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 607 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Jikun
authors.initials: J
authors.lastname: Li
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 607 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Weixue
authors.initials: W
authors.lastname: Wang
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 607 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Yi-Liang
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Shivani
authors.initials: S
authors.lastname: Amin
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 607 South Mathews Avenue, Urbana, IL 61801 ; Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801.
authors.forename: Eric
authors.initials: E
authors.lastname: Oldfield