ISU (eukaryotes) and IscU (prokaryotes) are a homologous family of proteins that appear to provide a platform for assembly of [2Fe-2S] centers prior to delivery to an apo target protein. The intermediate [2Fe-2S] ISU-bound cluster is formed by delivery of iron and sulfur to the apo ISU, with the latter delivered through an IscS-mediated reaction. The identity of the iron donor has thus far not been established. In this paper we demonstrate human frataxin to bind from six to seven iron ions. Iron binding to frataxin has been quantitated by iron-dependent fluorescence measurements [K(D)(Fe(3+)) approximately 11.7 microM; (K(D)(Fe(2+)) approximately 55.0 microM] and isothermal titration calorimetry (ITC) [K(D)(Fe(3+)) approximately 10.2 microM]. Enthalpies and entropies for ferric ion binding were determined from calorimetric measurements. Both fluorescence (K(D) 0.45 microM) and ITC measurements (K(D) 0.15 microM) demonstrate holo frataxin to form a complex with ISU with sub-micromolar binding affinities. Significantly, apo frataxin does not bind to ISU, suggesting an important role for iron in cross-linking the two proteins and/or stabilizing the structure of frataxin that is recognized by ISU. Holo frataxin is also shown to mediate the transfer of iron from holo frataxin to nucleation sites for [2Fe-2S] cluster formation on ISU. We have demonstrated elsewhere [J. Am. Chem. Soc. 2002, 124, 8774-8775] that this iron-bound form of ISU is viable for assembly of holo ISU, either by subsequent addition of sulfide or by NifS-mediated sulfur delivery. Provision of holo frataxin and inorganic sulfide is sufficient for cluster assembly in up to 70% yield. With NifS as a sulfur donor, yields in excess of 70% of holo ISU were obtained. Both UV-vis and CD spectroscopic characteristics were found to be consistent with those of previously characterized ISU proteins. The time course for cluster assembly was monitored from the 456 nm absorbance of holo ISU formed during the [2Fe-2S] cluster assembly reaction. A kinetic rate constant k(obs) approximately 0.075 min(-)(1) was determined with 100 microM ISU, 2.4 mM Na(2)S, and 40 microM holo frataxin in 50 mM Tris-HCl (pH 7.5) with 4.3 mM DTT. Similar rates were obtained for NifS-mediated sulfur delivery, consistent with iron release from frataxin as a rate-limiting step in the cluster assembly reaction.

The reactivity of two [peptide-Cu] complexes ([GGH-Cu](-) and [KGHK-Cu](+)) toward DNA cleavage has been quantitatively investigated. Neither complex promoted hydrolytic cleavage, but efficient oxidative cleavage was observed in the presence of a mild reducing agent (ascorbate) and dioxygen. Studies with scavengers of ROS confirmed hydrogen peroxide to be an obligatory diffusible intermediate. While oxidative cleavage of DNA was observed for Cu(2+)(aq) under the conditions used, the kinetics of cleavage and reaction products/pathway were distinct from those displayed by [peptide-Cu] complexes. DNA cleavage chemistry is mediated by the H(2)O-dependent pathway following C-4'H abstraction from the minor groove. Such a cleavage path also provides a ready explanation for the linearization reaction promoted by [KGHK-Cu](+). Kinetic activities and reaction pathways are compared to published results on other chemical nucleases. Both [peptide-Cu] complexes were found to display second-order kinetics, with rate constants k(2) approximately 39 and 93 M(-1) s(-1) for [GGH-Cu](-) and [KGHK-Cu](+), respectively. Neither complex displayed enzyme-like saturation behavior, consistent with the relatively low binding affinity and residence time expected for association with dsDNA, and the absence of a prereaction complex. However, the intrinsic activity of each is superior to other catalyst systems, as determined from relative k(2) or k(cat)/K(m) values. Linearization of DNA was observed for [KGHK-Cu](+) relative to [GGH-Cu](-), consistent with the increased positive charge and longer residency time on dsDNA.

Human ferrochelatase, a mitochondrial membrane-associated protein, catalyzes the terminal step of heme biosynthesis by insertion of ferrous iron into protoporphyrin IX. The recently solved x-ray structure of human ferrochelatase identifies a potential binding site for an iron donor protein on the matrix side of the homodimer. Herein we demonstrate Hs holofrataxin to be a high affinity iron binding partner for Hs ferrochelatase that is capable of both delivering iron to ferrochelatase and mediating the terminal step in mitochondrial heme biosynthesis. A general regulatory mechanism for mitochondrial iron metabolism is described that defines frataxin involvement in both heme and iron-sulfur cluster biosyntheses. In essence, the distinct binding affinities of holofrataxin to the target proteins, ferrochelatase (heme synthesis) and ISU (iron-sulfur cluster synthesis), allows discrimination between the two major iron-dependent pathways and facilitates targeted heme biosynthesis following down-regulation of frataxin.Frataxin is a nuclear-encoded protein that is targeted to the mitochondrial matrix. Reduced frataxin expression, a causative agent of the neurological disorder Friedreich ataxia, results in mitochondrial iron accumulation. Recent evidence has pointed to a functional role for frataxin in mitochondrial iron metabolism, including iron-sulfur cluster (1-5) and heme (6 -8) biosynthesis. We have reported earlier that frataxin serves as an iron donor to ISU, the iron-sulfur cluster scaffold protein (1). Isothermal titration calorimetry and fluorescence quenching experiments demonstrated human frataxin to bind 6 or 7 iron ions with K D ϳ10 -50 M for the isolated protein (1). Holofrataxin was further shown to bind to ISU with a K D ϳ0.15 M, and the functional viability of frataxin as an iron donor for assembly of the [2Fe-2S] cluster of ISU in the presence of a sulfur donor was demonstrated through kinetic and spectroscopic studies (1). Iron release by frataxin appeared to be the rate-limiting step. Overall these results correlate well with other published observations concerning a possible role for frataxin in iron-sulfur cluster biosynthesis (2-5).To further characterize potential roles for frataxin as a mitochondrial iron donor, we have investigated the involvement of Hs frataxin in cellular heme biosynthesis as an iron donor to Hs ferrochelatase. Although the identity of the iron donor protein in heme biosynthesis has not been established, involvement by frataxin has been suggested on the basis of yeast studies that demonstrated mitochondrial iron to be unavailable for heme biosynthesis in cells lacking frataxin (6 -8). Dancis and co-workers (6) have recently reported genetics experiments that implicate the involvement of yeast frataxin in heme biosynthesis and have estimated a binding affinity (K D ) for frataxin to ferrochelatase of ϳ40 nM by surface plasmon resonance, although no evidence for frataxin-mediated iron delivery in heme biosynthesis was presented. Herein we characterize the interaction of human f...

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Density functional theory and continuum dielectric methods have been employed to evaluate the free energy of successive aqua-substitution reactions:  [Mg(H2O)6- m - n L m L‘ n ]2+ mz + ny + L z → [Mg(H2O)5- m - n L m +1L‘ n ]2+( m +1) z + ny + H2O. The ligands L z or L‘ y under consideration are simple organic molecules that model the amino acid residues that are most commonly found as protein ligands to divalent magnesium. In addition, the Protein Data Bank was surveyed for 3-dimensional protein structures of magnesium-binding sites containing only amino acid ligands. The results obtained were used to delineate the most thermodynamically preferable inner-sphere coordination environment for magnesium. The results also suggest an explanation for two phenomena:  (i) the observed inner-sphere binding mode of Mg2+ to proteins and (ii) the unique role of Mg2+ as a carrier of water molecules that mediate enzymatic hydrolysis reactions.

Axial iron ligation and protein encapsulation of the heme cofactor have been investigated as effectors of the reduction potential (E degrees ') of cytochrome c through direct electrochemistry experiments. Our approach was that of partitioning the E degrees ' changes resulting from binding of imidazole, 2-methyl-imidazole, ammonia, and azide to both cytochrome c and microperoxidase-11 (MP11), into the enthalpic and entropic contributions. N-Acetylmethionine binding to MP11 was also investigated. These ligands replace Met80 and a water molecule axially coordinated to the heme iron in cytochrome c and MP11, respectively. This factorization was achieved through variable temperature E degrees ' measurements. In this way, we have found that (i) the decrease in E degrees ' of cytochrome c due to Met80 substitution by a nitrogen-donor ligand is almost totally enthalpic in origin, as a result of the stronger electron donor properties of the exogenous ligand which selectively stabilize the ferric state; (ii) on the contrary, the binding of the same ligands and N-acetylmethionine to MP11 results in an enthalpic stabilization of the reduced state, whereas the entropic effect invariably decreases E degrees ' (the former effect prevails for the methionine ligand and the latter for the nitrogenous ligands). A comparison of the reduction thermodynamics of cytochrome c and the MP11 adducts offers insight on the effect of changing axial heme ligation and heme insertion into the folded polypeptide chain. Principally, we have found that the overall E degrees ' increase of approximately 400 mV, comparing MP11 and native cytochrome c, consists of two opposite enthalpic and entropic terms of approximately +680 and -280 mV, respectively. The enthalpic term includes contributions from both axial methionine binding (+300 mV) and protein encapsulation of the heme (+380 mV), whereas the entropic term is almost entirely manifest at the stage of axial ligand binding. Both terms are dominated by the effects of water exclusion from the heme environment.

Glycosylated natural products such as bleomycin, neocarzinostatin, and calicheamicin γ 1 are efficient antitumor agents that cleave ds DNA by pathways that involve redox chemistry. In this paper we demonstrate the use of metalloderivatives of natural aminoglycosides as efficient DNA cleavage agents in the absence of external reducing agents. Kinetic characterization of DNA cleavage by copper neamine under Michaelis-Menten-"type" reaction conditions revealed a maximal reaction velocity V max ′ ) 0.031 min -1 , equivalent to a greater than 50-million-fold rate enhancement in DNA cleavage, when uncorrected for catalyst concentrations. Under true Michaelis conditions, a maximal reaction velocity V max ) 0.0595 min -1 was obtained (with k cat ) 5.95 × 10 -4 min -1 ), corresponding to a million-fold rate enhancement using micromolar concentrations of Cu 2+ -neamine. The specificity constants for DNA cleavage by copper neamine (k cat /K M ) 4.8 × 10 5 h -1 M -1 ) are 2 orders of magnitude greater than those reported elsewhere for synthetic compounds, at this time. Cleavage mediated by Cu 2+ -(kanamycin A) was found to be even more efficient. DNA cleavage was not inhibited by SOD, NaN 3 , DMSO, or EtOH, nor by handling under anaerobic conditions. The results of gel electrophoretic experiments provide clear evidence for a hydrolytic cleavage pathway with generation of 5′phosphate and 3′-hydroxyl termini.

A library of complexes that included iron, cobalt, nickel, and copper chelates of cyclam, cyclen, DOTA, DTPA, EDTA, tripeptide GGH, tetrapeptide KGHK, NTA, and TACN was evaluated for DNA nuclease activity, ascorbate consumption, superoxide and hydroxyl radical generation, and reduction potential under physiologically relevant conditions. Plasmid DNA cleavage rates demonstrated by combinations of each complex and biological coreactants were quantified by gel electrophoresis, yielding second-order rate constants for DNAsupercoiled to DNAnicked conversion up to 2.5 ×106 M-1min-1, and for DNAnicked to DNAlinear up to 7 ×105 M-1min-1. Relative rates of radical generation and characterization of radical species were determined by reaction with the fluorescent radical probe TEMPO-9-AC and rhodamine B. Ascorbate turnover rate constants ranging from 9.1×10-3 to 8.2 min-1 were determined, although many complexes demonstrated no measureable activity. Inhibition and Freifelder-Trumbo analysis of DNA cleavage supported concerted cleavage of dsDNA by a metal associated ROS in the case of Cu2+(aq), Cu-KGHK, Co-KGHK, and Cu-NTA and stepwise cleavage for Fe2+(aq), Cu-cyclam, Cu-cyclen, Co-cyclen, Cu-EDTA, Ni-EDTA, Co-EDTA, Cu-GGH, and Co-NTA. Reduction potentials varied over the range from -362 mV to +1111 mV versus NHE, and complexes demonstrated optimal catalytic activity in the range of the physiological redox coreactants ascorbate and peroxide (-66 to +380 mV).