Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer (ET) in photosynthesis and respiration to catalysis in industrial and fuel cell research. Nature accomplishes these functions by employing only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or ET properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centers1,2 or fuel cell catalysts3 for energy conversion. We have shown here that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin (Az) over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of Az is now predictable across the full range of cupredoxin potentials.
We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M ; V max ), EPR and 1 H, 2 H, 13 C, 31 P, and 57 Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57 Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π∕σ "metallacycle" or η 2 -alkenyl complex. The complex is poised to interact with H þ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η 1 -allyl complex. After reduction, this forms an η 3 -allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1 H, 2 H, and 13 C ENDOR, where hyperfine couplings of approximately 6 MHz for 13 C and 10 MHz for 1 H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe 4 S 4 -containing proteins, such as IspG.enzyme inhibition | iron-sulfur protein | isoprenoid biosynthesis | nonmevalonate pathway E nzymes that catalyze the formation of isoprenoids are of interest as drug targets. There are two main pathways involved in the early steps in isoprenoid biosynthesis: The mevalonate pathway found in animals and in pathogens such as Staphylococcus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative agents of staph infections, Chagas' disease and the leishmaniases), and the nonmevalonate or Rohmer pathway found in most pathogenic bacteria, as well as in the malaria parasite, Plasmodium falciparum (1). Both pathways lead to formation of the C 5 -isoprenoids isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2). In the later stages of isoprenoid biosynthesis, these C 5 -compounds then form the farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) used in protein prenylation, sterol, and carotenoid biosynthesis. Understanding how the enzymes catalyzing these "downstream" events function has led to a better understanding of e.g. how FPP synthase (2) and GGPP synthase function, and can be inhibited (3); the discovery that bisphosphonates have potent antiparasitic activity (4); the clinical use of amiodarone (a squalene oxidase and oxidosqualene cyclase inhibitor) against Chagas' disease (5; 6) and leishmaniasis (7); anticancer agents that inhibit both FPPS and GGPPS (8); as wel...
The one-electron oxidations of a series of diiron(I) dithiolato carbonyls were examined to evaluate the factors that affect the oxidation state assignments, structures, and reactivity of these lowmolecular weight models for the H ox state of the [FeFe]-hydrogenases. The propanedithiolates Fe 2 (S 2 C 3 H 6 )(CO) 3 (L)(dppv) (L = CO, PMe 3 , Pi-Pr 3 ) oxidize at potentials ~180 mV milder than the related ethanedithiolates (Angew. Chem. Int. Ed. 2007, 46, 6152). The steric clash between the central methylene of the propanedithiolate and the phosphine favors the rotated structure, which forms upon oxidation. EPR spectra for the mixed-valence cations indicate that the unpaired electron is localized on the Fe(CO)(dppv) center in both [Fe 2 (S 2 C 3 H 6 )(CO) 4 (dppv)]BF 4 and [Fe 2 (S 2 C 3 H 6 ) (CO) 3 (PMe 3 )(dppv)]BF 4 , as seen previously for the ethanedithiolate [Fe 2 (S 2 C 2 H 4 )(CO) 3 (PMe 3 ) (dppv)]BF 4 . For [Fe 2 (S 2 C n H 2n )(CO) 3 (Pi-Pr 3 )(dppv)]BF 4 , however, the spin is localized on the Fe (CO) 2 (Pi-Pr 3 ) center, although the Fe(CO)(dppv) site is rotated in the crystalline state. IR and EPR spectra, as well as redox potentials and DFT-calculations, suggest, however, that the Fe(CO) 2 (PiPr 3 ) site is rotated in solution, driven by steric factors. Analysis of the DFT-computed partial atomic charges for the mixed-valence species shows that the Fe atom featuring a vacant apical coordination position is an electrophilic Fe(I) center. One-electron oxidation of [Fe 2 (S 2 C 2 H 4 )(CN) (CO) 3 (dppv)] − resulted in 2e oxidation of 0.5 equiv to give the μ-cyano derivative [Fe I 2 (S 2 C 2 H 4 ) (CO) 3 (dppv)](μ-CN)[Fe II 2 (S 2 C 2 H 4 )(μ-CO)(CO) 2 (CN)(dppv)], which was characterized spectroscopically.
The nqr operon from Vibrio cholerae, encoding the entire six-subunit, membrane-associated, Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), was cloned under the regulation of the P(BAD) promoter. The enzyme was successfully expressed in V. cholerae. To facilitate molecular genetics studies of this sodium-pumping enzyme, a host strain of V. cholerae was constructed in which the genomic copy of the nqr operon was deleted. By using a vector containing a six-histidine tag on the carboxy terminus of the NqrF subunit, the last subunit in the operon, the recombinant enzyme was readily purified by affinity chromatography in a highly active form from detergent-solubilized membranes of V. cholerae. The recombinant enzyme has a high specific activity in the presence of sodium. NADH consumption was assessed at a turnover number of 720 electrons per second. When purified using dodecyl maltoside (DM), the isolated enzyme contains approximately one bound ubiquinone, whereas if the detergent LDAO is used instead, the quinone content of the isolated enzyme is negligible. Furthermore, the recombinant enzyme, purified with DM, has a relatively low rate of reaction with O(2) (10-20 s(-1)). In steady state turnover, the isolated, recombinant enzyme exhibits up to 5-fold stimulation by sodium and functions as a primary sodium pump, as reported previously for Na(+)()-NQR from other bacterial sources. When reconstituted into liposomes, the recombinant Na(+)-NQR generates a sodium gradient and a Delta Psi across the membrane. SDS-PAGE resolves all six subunits, two of which, NqrB and NqrC, contain covalently bound flavin. A redox titration of the enzyme, monitored by UV-visible spectroscopy, reveals three n = 2 redox centers and one n = 1 redox center, for which the presence of three flavins and a 2Fe-2S center can account. The V. cholerae Na(+)-NQR is well-suited for structural studies and for the use of molecular genetics techniques in addressing the mechanism by which NADH oxidation is coupled to the pumping of Na(+) across the membrane.
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