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The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe 2 ADT(CO) 3 (CN) 2 , where ADT represents the bridging ligand aza-propane-dithiolate. The terminal diatomic ligands all coordinate in a basal configuration, and one CO bridges the two irons leaving an open coordination site at which the hydrogen species and the competitive inhibitor CO bind. Externally supplied CO is expected to coordinate in an apical configuration. However, an alternative configuration has been proposed in which, due to ligand rotation, the CN - bound to the distal Fe becomes apical. Using selective 13 C isotope labeling of the CN - and CO ext ligands in combination with pulsed 13 C electron-nuclear-nuclear triple resonance spectroscopy, spin polarization effects are revealed that, according to density functional theory calculations, are consistent with only the "unrotated" apical CO ext configuration.

Human de novo iron-sulfur (Fe-S) assembly complex consists of cysteine desulfurase NFS1, accessory protein ISD11, acyl carrier protein ACP, scaffold protein ISCU, and allosteric activator frataxin (FXN). FXN binds the NFS1-ISD11-ACP-ISCU complex (SDAU), to activate the desulfurase activity and Fe-S cluster biosynthesis. In the absence of FXN, the NFS1-ISD11-ACP (SDA) complex was reportedly inhibited by binding of recombinant ISCU. Recent studies also reported a substitution at position Met141 on the yeast ISCU orthologue Isu, to Ile, Leu, Val, or Cys, could bypass the requirement of FXN for Fe-S cluster biosynthesis and cell viability. Here, we show that recombinant human ISCU binds zinc(II) ion, as previously demonstrated with the E. coli orthologue IscU. Surprisingly, the relative proportion between zinc-bound and zinc-depleted forms varies among purification batches. Importantly the presence of zinc in ISCU impacts SDAU desulfurase activity. Indeed, removal of zinc(II) ion from ISCU causes a moderate but significant increase in activity compared to SDA alone, and FXN can activate both zinc-depleted and zinc-bound forms of ISCU complexed to SDA. Taking into consideration the inhibition of desulfurase activity by zinc-bound ISCU, we characterized wild type ISCU and the M140I, M140L, and M140V variants under both zinc-bound and zinc-depleted conditions, and did not observe significant differences in the biochemical and biophysical properties between wild-type and variants. Importantly, in the absence of FXN, ISCU variants behaved like wild-type and did not stimulate the desulfurase activity of the SDA complex. This study therefore identifies an important regulatory role for zinc-bound ISCU in modulation of the human Fe-S assembly system in vitro and reports no 'FXN bypass' effect on mutations at position Met140 in human ISCU. Furthermore, this study also calls for caution in interpreting studies involving recombinant ISCU by taking into consideration the influence of the bound zinc(II) ion on SDAU complex activity., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Mitochondrial DNA (mtDNA) mutations become more prevalent with age and are postulated to contribute to the ageing process. Point mutations of mtDNA have been suggested to originate from two main sources, i.e. replicative errors and oxidative damage, but the contribution of each of these processes is much discussed. To elucidate the origin of mtDNA mutations, we measured point mutation load in mice with deficient mitochondrial base-excision repair (BER) caused by knockout alleles preventing mitochondrial import of the DNA repair glycosylases OGG1 and MUTYH (Ogg1 dMTS, Mutyh dMTS). Surprisingly, we detected no increase in the mtDNA mutation load in old Ogg1 dMTS mice. As DNA repair is especially important in the germ line, we bred the BER deficient mice for five consecutive generations but found no increase in the mtDNA mutation load in these maternal lineages. To increase reactive oxygen species (ROS) levels and oxidative damage, we bred the Ogg1 dMTS mice with tissue specific Sod2 knockout mice. Although increased superoxide levels caused a plethora of changes in mitochondrial function, we did not detect any changes in the mutation load of mtDNA or mtRNA. Our results show that the importance of oxidative damage as a contributor of mtDNA mutations should be re-evaluated.

The effects of cyanide on enzymatic activity and absorption spectra in the visible and mid-IR (2150-1850cm -1 ) regions were characterized for purified HydSL hydrogenase from the purple sulfur bacterium Thiocapsa (T.) roseopersicina BBS. Prolonged incubation (over hours) of T. roseopersicina hydrogenase with exogenous cyanide was shown to result in an irreversible loss of activity of the enzyme in both the oxidized (as isolated) and H 2 -reduced states. The frequency position of the active site CO and CN - ligand stretching bands in the Fourier transform infrared (FTIR) spectrum of the oxidized form of hydrogenase was not influenced by cyanide treatment. The 410-nm absorption band characteristic of hydrogenase iron‑sulfur clusters showed a bleaching concomitantly with cyanide inactivation. A new band at 2038cm -1 was present in the FTIR spectrum of the cyanide-inactivated preparation, which band is assignable to ferrocyanide as a possible product of a destructive interaction of hydrogenase with cyanide. The results are interpreted in terms of a slow destruction of iron‑sulfur clusters of hydrogenase in the presence of cyanide accompanied by a release of iron ions in the form of ferrocyanide into the surrounding solution. Such a slow and irreversible cyanide-dependent inactivation seems to be complementary to a recently described rapid, reversible inhibitory reaction of cyanide with the active site of hydrogenases [S.V. Hexter, M.-W. Chung, K.A. Vincent, F.A. Armstrong, J. Am. Chem. Soc. 136 (2014) 10470-10477]., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H 2 ) production and H 2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H 2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al. Nat. Chem. 2014, 4, 336-342] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect.

The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively (57)Fe-labeled binuclear subsite is described. The precursor [(57)Fe2(adt)(CN)2(CO)4](2-) was synthesized from the (57)Fe metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)2[(57)Fe2(adt)(CN)2(CO)4] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]H subcluster. Complementary (57)Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with (57)FeCl3 and Na2S. The Hox-CO state of [2(57)Fe]H and [4(57)Fe-4S]H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy.

A computational mechanistic model of superoxide (O2•-) formation in the mitochondrial electron transport chain (ETC) was developed to facilitate the quantitative analysis of factors controlling mitochondrial O2•- production and assist in the interpretation of experimental studies. The model takes into account all individual electron transfer reactions in Complexes I and III. The model accounts for multiple, often seemingly contradictory observations on the effects of ΔΨ and ΔpH, and for the effects of multiple substrate and inhibitor conditions, including differential effects of Complex III inhibitors antimycin A, myxothiazol and stigmatellin. Simulation results confirm that, in addition to O2•- formation in Complex III and at the flavin site of Complex I, the quinone binding site of Complex I is an additional superoxide generating site that accounts for experimental observations on O2•- production during reverse electron transfer. However, our simulation results predict that, when cytochrome c oxidase is inhibited during oxidation of succinate, ROS production at this site is eliminated and almost all superoxide in Complex I is generated by reduced FMN, even when the redox pressure for reverse electron transfer from succinate is strong. In addition, the model indicates that conflicting literature data on the kinetics of electron transfer in Complex III involving the iron-sulfur protein-cytochrome bL complex can be resolved in favor of a dissociation of the protein only after electron transfer to cytochrome bH. The model predictions can be helpful in understanding factors driving mitochondrial superoxide formation in intact cells and tissues., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Oxygen activation at the active sites of [FeFe] hydrogenases has been proposed to be the initial step of irreversible oxygen-induced inhibition of these enzymes. On the basis of a first theoretical study into the thermodynamics of O2 activation [Inorg. Chem. 2009, 48, 7127] we here investigate the kinetics of possible reaction paths at the distal iron atom of the active site by means of density functional theory. A sequence of steps is proposed to either form a reactive oxygen species (ROS) or fully reduce O2 to water. In this reaction cascade, two branching points are identified where water formation directly competes with harmful oxygen activation reactions. The latter are water formation by O-O bond cleavage of a hydrogen peroxide-bound intermediate competing with H2O2 dissociation and CO2 formation by a putative iron-oxo species competing with protonation of the iron-oxo species to form a hydroxyo ligand. Furthermore, we show that proton transfer to activated oxygen is fast and that proton supply to the active site is vital to prevent ROS dissociation. If sufficiently many reduction equivalents are available, oxygen activation reactions are accelerated, and oxygen reduction to water becomes possible.

The plastid of the malaria parasite, the apicoplast, is essential for parasite survival. It houses several pathways of bacterial origin that are considered attractive sites for drug intervention. Among these is the sulfur mobilization (SUF) pathway of Fe-S cluster biogenesis. Although the SUF pathway is essential for apicoplast maintenance and parasite survival, there has been limited biochemical investigation of its components and inhibitors of Plasmodium SUFs have not been identified. We report the characterization of two proteins, Plasmodium falciparum SufS (PfSufS) and PfSufE, that mobilize sulfur in the first step of Fe-S cluster assembly and confirm their exclusive localization to the apicoplast. The cysteine desulfurase activity of PfSufS is greatly enhanced by PfSufE, and the PfSufS-PfSufE complex is detected in vivo. Structural modeling of the complex reveals proximal positioning of conserved cysteine residues of the two proteins that would allow sulfide transfer from the PLP (pyridoxal phosphate) cofactor-bound active site of PfSufS. Sulfide release from the l-cysteine substrate catalyzed by PfSufS is inhibited by the PLP inhibitor d-cycloserine, which forms an adduct with PfSufS-bound PLP. d-Cycloserine is also inimical to parasite growth, with a 50% inhibitory concentration close to that reported for Mycobacterium tuberculosis, against which the drug is in clinical use. Our results establish the function of two proteins that mediate sulfur mobilization, the first step in the apicoplast SUF pathway, and provide a rationale for drug design based on inactivation of the PLP cofactor of PfSufS., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Enzymes of the methylerythritol phosphate pathway of isoprenoid biosynthesis are attractive anti-infective drug targets. The last two enzymes of this pathway, IspG and IspH, are [Fe4 S4 ] proteins that are not produced by humans and catalyze 2 H(+) / 2 e(-) reductions with novel mechanisms. In this Review, we summarize recent advances in structural, mechanistic, and inhibitory studies of these two enzymes. In particular, mechanistic proposals involving bioorganometallic intermediates are presented, and compared with other mechanistic possibilities. In addition, inhibitors based on substrate analogues as well as developed by rational design and compound-library screening, are discussed. The results presented support bioorganometallic catalytic mechanisms for IspG and IspH, and open up new routes to anti-infective drug design targeting [Fe4 S4 ] clusters in proteins., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nature is a valuable source of inspiration in the design of catalysts, and various approaches are used to elucidate the mechanism of hydrogenases, the enzymes that oxidize or produce H2. In FeFe hydrogenases, H2 oxidation occurs at the H-cluster, and catalysis involves H2 binding on the vacant coordination site of an iron centre. Here, we show that the reversible oxidative inactivation of this enzyme results from the binding of H2 to coordination positions that are normally blocked by intrinsic CO ligands. This flexibility of the coordination sphere around the reactive iron centre confers on the enzyme the ability to avoid harmful reactions under oxidizing conditions, including exposure to O2. The versatile chemistry of the diiron cluster in the natural system might inspire the design of novel synthetic catalysts for H2 oxidation.

Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.

Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in vivo NAD biosynthesis inhibiting activity in E. coli. The inhibitory effect can be explained by the coordination of DTHPA thiolate groups to a unique Fe site of the NadA [4Fe-4S] cluster., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Designing O(2)-tolerant hydrogenases is a major challenge in applying [Fe-Fe]H(2)ases for H(2) production. The inhibition involves transport of oxygen through the enzyme to the H-cluster, followed by binding and subsequent deactivation of the active site. To explore the nature of the oxygen diffusion channel for the hydrogenases from Desulfovibrio desulfuricans (Dd) and Clostridium pasteurianum (Cp), empirical molecular dynamics simulations were performed. The dynamic nature of the oxygen pathways in Dd and Cp was elucidated, and insight is provided, in part, into the experimental observation on the difference of oxygen inhibition in Dd and the hydrogenase from Clostridium acetobutylicum (Ca, assumed homologous to Cp). Further, to gain an understanding of the mechanism of oxygen inhibition of the [Fe-Fe]H(2)ase, density functional theory calculations of model compounds composed of the H-cluster and proximate amino acids are reported. Confirmation of the experimentally based suppositions on inactivation by oxygen at the [2Fe](H) domain is provided, validating the model compounds used and oxidation state assumptions, further explaining the mode of damage. This unified approach provides insight into oxygen diffusion in the enzyme, followed by deactivation at the H-cluster.

The anaerobic global regulator FNR from Escherichia coli is a [4Fe-4S](2+) cluster-containing dimer that is inactivated by O(2) through disruption of the Fe-S cluster and conversion to the monomeric apoprotein. It was shown that apo-FNR is subject to ClpXP proteolysis, and two recognition sites, amino acids 5-11 and amino acids 249 and 250, are responsible for targeting FNR to the protease. However, how the exposure of these sites is mediated such that only apo-FNR is recognized by the ClpXP protease and is degraded in a regulated manner so that a sufficient and similar FNR level is maintained in both anaerobic and aerobic conditions is unknown. To investigate this, we performed three-alanine scanning on amino acids 2-19 and 236-250 that are in the proximity of the two ClpXP recognition sites, and their functions remain unknown. We found that three-alanine substitution of residues 239-241 (LAQ239-241A(3)) and 242-244 (LAG242-244A(3)) caused reduced FNR protein levels, transcription activities, and growth rates under anaerobic conditions. In vivo degradation assays demonstrated that these mutants were degraded significantly faster than the wild type (WT), and either deletion of clpXP or blocking the ClpXP recognition site of amino acids 249 and 250 stabilizes these proteins. Circular dichroism analysis revealed that introduction of LAQ239-241A(3) caused conformational changes with a significant loss of secondary structures in both WT and an O(2) stable FNR dimer, FNR D154A. We propose that the region of amino acids 239-244 plays a negative role in the proteolysis of FNR by promoting a structural fold that limits the exposure of the proximal ClpXP site to the protease.

Members of the bacterial and mitochondrial iron-sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, respectively. An additional protein, Iba57, physically interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technology. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.

Zinc pyrithione (ZPT) is an antimicrobial material with widespread use in antidandruff shampoos and antifouling paints. Despite decades of commercial use, there is little understanding of its antimicrobial mechanism of action. We used a combination of genome-wide approaches (yeast deletion mutants and microarrays) and traditional methods (gene constructs and atomic emission) to characterize the activity of ZPT against a model yeast, Saccharomyces cerevisiae. ZPT acts through an increase in cellular copper levels that leads to loss of activity of iron-sulfur cluster-containing proteins. ZPT was also found to mediate growth inhibition through an increase in copper in the scalp fungus Malassezia globosa. A model is presented in which pyrithione acts as a copper ionophore, enabling copper to enter cells and distribute across intracellular membranes. This is the first report of a metal-ligand complex that inhibits fungal growth by increasing the cellular level of a different metal.

Biotin synthase catalyzes the conversion of dethiobiotin (DTB) to biotin through the oxidative addition of sulfur between two saturated carbon atoms, generating a thiophane ring fused to the existing ureido ring. Biotin synthase is a member of the radical SAM superfamily, composed of enzymes that reductively cleave S-adenosyl-l-methionine (SAM or AdoMet) to generate a 5'-deoxyadenosyl radical that can abstract unactivated hydrogen atoms from a variety of organic substrates. In biotin synthase, abstraction of a hydrogen atom from the C9 methyl group of DTB would result in formation of a dethiobiotinyl methylene carbon radical, which is then quenched by a sulfur atom to form a new carbon-sulfur bond in the intermediate 9-mercaptodethiobiotin (MDTB). We have proposed that this sulfur atom is the μ-sulfide of a [2Fe-2S](2+) cluster found near DTB in the enzyme active site. In the present work, we show that formation of MDTB is accompanied by stoichiometric generation of a paramagnetic FeS cluster. The electron paramagnetic resonance (EPR) spectrum is modeled as a 2:1 mixture of components attributable to different forms of a [2Fe-2S](+) cluster, possibly distinguished by slightly different coordination environments. Mutation of Arg260, one of the ligands to the [2Fe-2S] cluster, causes a distinctive change in the EPR spectrum. Furthermore, magnetic coupling of the unpaired electron with (14)N from Arg260, detectable by electron spin envelope modulation (ESEEM) spectroscopy, is observed in WT enzyme but not in the Arg260Met mutant enzyme. Both results indicate that the paramagnetic FeS cluster formed during catalytic turnover is a [2Fe-2S](+) cluster, consistent with a mechanism in which the [2Fe-2S](2+) cluster simultaneously provides and oxidizes sulfide during carbon-sulfur bond formation., (© 2011 American Chemical Society)

Iron-sulfur clusters are versatile electron transfer cofactors, ubiquitous in metalloenzymes such as hydrogenases. In the oxygen-tolerant Hydrogenase I from Aquifex aeolicus such electron "wires" form a relay to a diheme cytb, an integral part of a respiration pathway for the reduction of O(2) to water. Amino acid sequence comparison with oxygen-sensitive hydrogenases showed conserved binding motifs for three iron-sulfur clusters, the nature and properties of which were unknown so far. Electron paramagnetic resonance spectra exhibited complex signals that disclose interesting features and spin-coupling patterns; by redox titrations three iron-sulfur clusters were identified in their usual redox states, a [3Fe4S] and two [4Fe4S], but also a unique high-potential (HP) state was found. On the basis of (57)Fe Mössbauer spectroscopy we attribute this HP form to a superoxidized state of the [4Fe4S] center proximal to the [NiFe] site. The unique environment of this cluster, characterized by a surplus cysteine coordination, is able to tune the redox potentials and make it compliant with the [4Fe4S](3+) state. It is actually the first example of a biological [4Fe4S] center that physiologically switches between 3+, 2+, and 1+ oxidation states within a very small potential range. We suggest that the (1 + /2+) redox couple serves the classical electron transfer reaction, whereas the superoxidation step is associated with a redox switch against oxidative stress.

Dihydrogen (H(2)) production by [FeFe]-hydrogenases is strongly inhibited by formaldehyde (methanal) in a reaction that is rapid, reversible, and specific to this type of hydrogenase. This discovery, using three [FeFe]-hydrogenases that are homologous about the active site but otherwise structurally distinct, was made by protein film electrochemistry, which measures the activity (as electrical current) of enzymes immobilized on an electrode; importantly, the inhibitor can be removed after addition. Formaldehyde causes rapid loss of proton reduction activity which is restored when the solution is exchanged. Inhibition is confirmed by conventional solution assays. The effect depends strongly on the direction of catalysis: inhibition of H(2) oxidation is much weaker than for H(2) production, and formaldehyde also protects against CO and O(2) inactivation. By contrast, inhibition of [NiFe]-hydrogenases is weak. The results strongly suggest that formaldehyde binds at, or close to, the active site of [FeFe]-hydrogenases at a site unique to this class of enzyme--highly conserved lysine and cysteine residues, the bridgehead atom of the dithiolate ligand, or the reduced Fe(d) that is the focal center of catalysis.

[Fe]-Hydrogenase catalyzes the reversible activation of H(2). CO and CN(-) inhibit this enzyme with low affinity (K(i)≅0.1 mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN(-), strongly inhibit [Fe]-hydrogenase (K(i) as low as 1 nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV-Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

We report the inhibition of the Aquifex aeolicus IspH enzyme (LytB, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, EC 1.17.1.2) by a series of diphosphates and bisphosphonates. The most active species was an alkynyl diphosphate having IC(50) = 0.45 microM (K(i) approximately 60 nM), which generated a very large change in the 9 GHz EPR spectrum of the reduced protein. On the basis of previous work on organometallic complexes, together with computational docking and quantum chemical calculations, we propose a model for alkyne inhibition involving pi (or pi/sigma) "metallacycle" complex formation with the unique fourth Fe in the Fe(4)S(4) cluster. Aromatic species had less activity, and for these we propose an inhibition model based on an electrostatic interaction with the active site E126. Overall, the results are of broad general interest since they not only represent the first potent IspH inhibitors but also suggest a conceptually new approach to inhibiting other Fe(4)S(4)-cluster-containing proteins that are of interest as drug and herbicide targets.

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.

Hydrogenases comprise a variety of enzymes that catalyze the reversible oxidation of molecular hydrogen. Out of this group, [FeFe]-hydrogenase shows the highest activity for hydrogen production which is, therefore, of great interest in the field of renewable energies. Unfortunately, this comes with the flaw of a generally very high sensitivity against molecular oxygen that irreversibly inhibits this enzyme. While many studies have already addressed the mechanism of hydrogen formation by [FeFe]-hydrogenase, little is known about the molecular and mechanistic details leading to enzyme inactivation by O(2). In order to elucidate this process, we performed density functional theory calculations on several possible O(2) adducts of the catalytic center--the so-called H-cluster--and show that the direct interaction of the [2Fe](H) subsite with dioxygen is an exothermic and specific reaction in which O(2) most favorably binds in an end-on manner to the distal Fe(d). Based on the results, we propose a protonation mechanism that can explain the irreversibility of dioxygen-induced enzyme inactivation by water release and degradation of the ligand environment of the H-cluster.

The [FeFe] hydrogenase (CrHydA1) of the green alga Chlamydomonas reinhardtii is the smallest hydrogenase known and can be considered as a "minimal unit" for biological H(2) production. Due to the absence of additional FeS clusters as found in bacterial [FeFe] hydrogenases, it was possible to specifically study its catalytic iron-sulfur cluster (H-cluster) by X-ray absorption spectroscopy (XAS) at the Fe K-edge. The XAS analysis revealed that the CrHydA1 H-cluster consists of a [4Fe4S] cluster and a diiron site, 2Fe(H), which both are similar to their crystallographically characterized bacterial counterparts. Determination of the individual Fe-Fe distances in the [4Fe4S] cluster ( approximately 2.7 A) and in the 2Fe(H) unit ( approximately 2.5 A) was achieved. Fe-C( horizontal lineO/N) and Fe-S bond lengths were in good agreement with crystallographic data on bacterial enzymes. The loss of Fe-Fe distances in protein purified under mildly oxidizing conditions indicated partial degradation of the H-cluster. Bond length alterations detected after incubation of CrHydA1 with CO and H(2) were related to structural and oxidation state changes at the catalytic Fe atoms, e.g., to the binding of an exogenous CO at 2Fe(H) in CO-inhibited enzyme. Our XAS studies pave the way for the monitoring of atomic level structural changes at the H-cluster during H(2) catalysis.

The tellurium oxyanion tellurite is toxic for most organisms and it seems to alter a number of intracellular targets. In this work the toxic effects of tellurite upon Escherichia coli [4Fe-4S] cluster-containing dehydratases was studied. Reactive oxygen species (ROS)-sensitive fumarase A (FumA) and aconitase B (AcnB) as well as ROS-resistant fumarase C (FumC) and aconitase A (AcnA) were assayed in cell-free extracts from tellurite-exposed cells in both the presence and absence of oxygen. While over 90 % of FumA and AcnB activities were lost in the presence of oxygen, no enzyme inactivation was observed in anaerobiosis. This result was not dependent upon protein biosynthesis, as determined using translation-arrested cells. Enzyme activity of purified FumA and AcnB was inhibited when exposed to an in vitro superoxide-generating, tellurite-reducing system (ITRS). No inhibitory effect was observed when tellurite was omitted from the ITRS. In vivo and in vitro reconstitution experiments with tellurite-damaged FumA and AcnB suggested that tellurite effects involve [Fe-S] cluster disabling. In fact, after exposing FumA to ITRS, released ferrous ion from the enzyme was demonstrated by spectroscopic analysis using the specific Fe(2+) chelator 2,2'-bipyridyl. Subsequent spectroscopic paramagnetic resonance analysis of FumA exposed to ITRS showed the characteristic signal of an oxidatively inactivated [3Fe-4S](+) cluster. These results suggest that tellurite inactivates enzymes of this kind via a superoxide-dependent disabling of their [4Fe-4S] catalytic clusters.

Excess copper is poisonous to all forms of life, and copper overloading is responsible for several human pathologic processes. The primary mechanisms of toxicity are unknown. In this study, mutants of Escherichia coli that lack copper homeostatic systems (copA cueO cus) were used to identify intracellular targets and to test the hypothesis that toxicity involves the action of reactive oxygen species. Low micromolar levels of copper were sufficient to inhibit the growth of both WT and mutant strains. The addition of branched-chain amino acids restored growth, indicating that copper blocks their biosynthesis. Indeed, copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster enzyme in this pathway. Other enzymes in this iron-sulfur dehydratase family were similarly affected. Inactivation did not require oxygen, in vivo or with purified enzyme. Damage occurred concomitant with the displacement of iron atoms from the solvent-exposed cluster, suggesting that Cu(I) damages these proteins by liganding to the coordinating sulfur atoms. Copper efflux by dedicated export systems, chelation by glutathione, and cluster repair by assembly systems all enhance the resistance of cells to this metal.

Biotin synthase (BioB) is an S-adenosylmethionine radical enzyme that catalyzes addition of sulfur to dethiobiotin to form the biotin thiophane ring. In vitro, Escherichia coli BioB is active for only one turnover, during which the [2Fe-2S]2+ cluster is destroyed, one sulfide from the cluster is incorporated as the biotin thiophane sulfur, while Fe2+ ions and the remaining S2- ion are released from the protein. The present work examines the fate of the protein following the loss of the FeS clusters. We examine the quaternary structure and thermal stability of active and inactive states of BioB, and find that loss of either the [4Fe-4S]2+ or [2Fe-2S]2+ clusters results in destabilization but not global unfolding of BioB. Using susceptibility to limited proteolysis as a guide, we find that specific regions of the protein appear to be transiently unfolded following loss of these clusters. We also examine the in vivo degradation of biotin synthase during growth in low-iron minimal media and find that BioB is degraded by an apparent ATP-dependent proteolysis mechanism that sequentially cleaves small fragments starting at the C-terminus. BioB appears to be resistant to degradation and capable of multiple turnovers only under high-iron conditions that favor repair of the FeS clusters, a process most likely mediated by the Isc or Suf iron-sulfur cluster assembly systems.

Mitochondrial complex II is a tumor suppressor comprised of four subunits (SdhA, SdhB, SdhC, and SdhD). Mutations in any of these should disrupt complex II enzymatic activity, yet defects in SdhA produce bioenergetic deficiency while defects in SdhB, SdhC, or SdhD induce tumor formation. The mechanisms underlying these differences are not known. We show that the inhibition of distal subunits of complex II, either pharmacologically or via RNA interference of SdhB, increases normoxic reactive oxygen species (ROS) production, increases hypoxia-inducible factor alpha (HIF-alpha) stabilization in an ROS-dependent manner, and increases growth rates in vitro and in vivo without affecting hypoxia-mediated activation of HIF-alpha. Proximal pharmacologic inhibition or RNA interference of complex II at SdhA, however, does not increase normoxic ROS production or HIF-alpha stabilization and results in decreased growth rates in vitro and in vivo. Furthermore, the enhanced growth rates resulting from SdhB suppression are inhibited by the suppression of HIF-1alpha and/or HIF-2alpha, indicating that the mechanism of SdhB-induced tumor formation relies upon ROS production and subsequent HIF-alpha activation. Therefore, differences in ROS production, HIF proliferation, and cell proliferation contribute to the differences in tumor phenotype in cells lacking SdhB as opposed to those lacking SdhA.

Cobalt is toxic for cells, but mechanisms of this toxicity are largely unknown. The biochemical and genetic experiments reported here demonstrate that iron-sulfur proteins are greatly affected in cobalt-treated Escherichia coli cells. Exposure of a wild-type strain to intracellular cobalt results in the inactivation of three selected iron-sulfur enzymes, the tRNA methylthio-transferase, aconitase, and ferrichrome reductase. Consistently, mutant strains lacking the [Fe-S] cluster assembly SUF machinery are hypersensitive to cobalt. Last, expression of iron uptake genes is increased in cells treated with cobalt. In vitro studies demonstrated that cobalt does not react directly with fully assembled [Fe-S] clusters. In contrast, it reacts with labile ones present in scaffold proteins (IscU, SufA) involved in iron-sulfur cluster biosynthesis. We propose a model wherein cobalt competes out iron during synthesis of [Fe-S] clusters in metabolically essential proteins.

[FeFe]-hydrogenases harbor a {2Fe3S} assembly bearing two CO and two CN- groups, a mu-CO ligand, and a vacant coordination site trans to the mu-CO group. Recent theoretical results obtained studying the isolated {2Fe3S} subsite indicated that one of the CN- ligands can easily move from the crystallographic position to the coordination site trans to the mu-CO group; such an isomerization would have a major impact on substrates and inhibitors binding regiochemistry and, consequently, on the catalytic mechanism. To shed light on this crucial issue, we have carried out hybrid QM/MM and free energy perturbation calculations on the whole enzyme, which demonstrate that the protein environment plays a crucial role and maintains the CN- group fixed in the position observed in the crystal structure; these results strongly support the hypothesis that the vacant coordination site trans to the mu-CO group has a crucial functional relevance both in the context of CO-mediated inhibition of the enzyme and in dihydrogen oxidation/evolution catalysis.

Tricarballylate, a citrate analogue, is considered the causative agent of grass tetany, a ruminant disease characterized by acute magnesium deficiency. Although the normal rumen flora cannot catabolize tricarballylate, the Gram-negative enterobacterium Salmonella enterica can. An operon dedicated to tricarballylate utilization (tcuABC) present in this organism encodes all functions required for tricarballylate catabolism. Tricarballylate is converted to the cis-aconitate in a single oxidative step catalyzed by the FAD-dependent tricarballylate dehydrogenase (TcuA) enzyme. We hypothesized that the uncharacterized TcuB protein was required to reoxidize the flavin cofactor in vivo. Here, we report the initial biochemical characterization of TcuB. TcuB is associated with the cell membrane and contains two 4Fe-4S clusters and heme. Site-directed mutagenesis of cysteinyl residues putatively required as ligands of the 4Fe-4S clusters completely inactivated TcuB function. TcuB greatly increased the Vmax of the TcuA reaction from 69 +/- 2 to 8200 +/- 470 nmol min-1 mg-1; the Km of TcuA for tricarballylate was unaffected. Inhibition of TcuB activity by an inhibitor of ubiquinone oxidation, 2,5-dibromo-3-methyl-6-isoproylbenzoquinone (DBMIB), implicated the quinone pool as the ultimate acceptor of electrons from FADH2. We propose a model for the electron flow from FADH2, to the 4Fe-4S clusters, to the heme, and finally to the quinone pool.

A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that the P. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.

Studies of the catalytic properties of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans by protein film voltammetry, under a H2 atmosphere, reveal and establish a variety of interesting properties not observed or measured quantitatively with other techniques. The catalytic bias (inherent ability to oxidize hydrogen vs reduce protons) is quantified over a wide pH range: the enzyme is proficient at both H2 oxidation (from pH > 6) and H2 production (pH < 6). Hydrogen production is inhibited by H2, but the effect is much smaller than observed for [NiFe]-hydrogenases from Allochromatium vinosum or Desulfovibrio fructosovorans. Under anaerobic conditions and positive potentials, the [FeFe]-hydrogenase is oxidized to an inactive form, inert toward reaction with CO and O2, that rapidly reactivates upon one-electron reduction under 1 bar of H2. The potential dependence of this interconversion shows that the oxidized inactive form exists in two pH-interconvertible states with pK(ox) = 5.9. Studies of the CO-inhibited enzyme under H2 reveals a strong enhancement of the rate of activation by white light at -109 mV (monitoring H2 oxidation) that is absent at low potential (-540 mV, monitoring H+ reduction), thus demonstrating photolability that is dependent upon the oxidation state.

The active form of the oxygen sensor fumarate nitrate reductase regulator (FNR) of Escherichia coli contains a [4Fe-4S] cluster which is converted to a [2Fe-2S] cluster after reaction with air, resulting in inactivation of FNR. Reaction of reconstituted [4Fe-4S].FNR with air resulted within 5 min in conversion to apoFNR. The rate was comparable to the rate known for [4Fe-4S].FNR/[2Fe-2S].FNR cluster conversion, suggesting that apoFNR is a product of [2Fe-2S].FNR decomposition and a final form of air-inactivated FNR in vitro. Formation of apoFNR and the redox state of the cysteinyl residues were determined in vitro by alkylation. FNR contains five cysteinyl residues, four of which (Cys20, Cys23, Cys29 and Cys122) ligate the FeS clusters. Alkylated FNR and proteolytic fragments thereof were analyzed by MALDI-TOF. ApoFNR formed by air inactivation of [4Fe-4S].FNR in vitro contained one or two disulfides. Only disulfide pairs Cys16/20 and Cys23/29 were formed; Cys122 was never part of a disulfide. The same type of disulfide was found in apoFNR obtained during isolation of FNR, suggesting that cysteine disulfide formation follows a fixed pattern. ApoFNR, including the form with two disulfides, can be reconstituted to [4Fe-4S].FNR after disulfide reduction. The experiments suggest that apoFNR is a major form of FNR under oxic conditions.

Iron-sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E(m) values) play a major role in defining the rate at which electrons are shuttled. The E(m) values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1-FS4) with E(m) values between -50 and -330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH(2)) binding, and protein control of its E(m). Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB-DmsC interface and are critical for the binding of the MQH(2) inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH(2) to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin-spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4([3Fe)(-)(4S])). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E(m) of FS4([3Fe)(-)(4S]) from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4([3Fe)(-)(4S]) lower its E(m), but mutations of negatively charged residues have negligible effects. The E(m) of FS4([3Fe)(-)(4S]) in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4([3Fe)(-)(4S]) E(m) of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E(m) for FS4([3Fe)(-)(4S]) of DmsB is approximately 275 mV.

IscU functions as a scaffold for Fe-S cluster assembly and transfer, and is known to be a substrate protein for molecular chaperones. Kinetic studies of Fe-S cluster transfer from holo IscU to apo Fd in the presence of chaperone DnaK demonstrate an inhibitory effect on the rate of Fe-S cluster transfer from IscU. Binding of DnaK reduces the rate of formation of the IscU-Fd complex (greater than 8-fold), but has little influence on the intrinsic rate of iron-sulfur cluster transfer to apo Fd. Apparently the molecular chaperone DnaK does not facilitate the process of Fe-S cluster transfer from IscU. Rather, DnaK has a modest influence on the stability of the IscU-bound Fe-S cluster that may reflect a more important role in promoting cluster assembly. In accord with prior observations the cochaperone DnaJ stimulates the ATPase activity of DnaK, but has a minimal influence on IscU cluster transfer activity, either alone or in concert with DnaK.

The orientation of the g-tensors of the Rieske iron-sulfur protein subunit was determined in a single crystal of the bovine mitochondrial cytochrome bc1 complex with stigmatellin in the Qo quinol binding site. The g-tensor principal axes are skewed with respect to the Fe-Fe and S-S atom direction in the 2Fe2S cluster, which is allowed by the lack of rigorous symmetry of the cluster. The asymmetric unit in the crystal is the active dimer, and the g-tensor axes have slightly different orientations relative to the iron-sulfur cluster in the two halves of the dimer. The g approximately 1.79 axis makes an average angle of 30 degrees with respect to the Fe-Fe direction and the g approximately 2.024 axis an average angle of 26 degrees with respect to the S-S direction. This assignment of the g-tensor axis directions indicates that conformations of the Rieske protein are likely the same in the cytochrome bc1 and b6f complexes and that the extent of motion of the Rieske head domain during the catalytic cycle has been highly conserved during evolution of these distantly related complexes.

It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O(2)(-)(*) or hydrogen peroxide (H(2)O(2)) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H(2)O(2). Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H(2)O(2) does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H(2)O(2). Nevertheless, prolonged exposure of mitochondria to steady-state levels of H(2)O(2) or O(2)(-)(*) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.

Mitochondria play a pivotal role as an ATP generator in aerobically growing cells, and their defects have long been implicated in the cellular aging process, although its detailed underlying mechanisms remain unclear. Recently, we found that, in the cellular senescent process of Chang cells induced by desferroxamine mesylate, an iron chelator, a significant decrease of intracellular ATP level was accompanied by decline in complex II activity, which preceded acquisition of the senescent phenotype. In the present study, we investigated the mechanism of how the mitochondrial ATP productivity was damaged by iron chelation and how complex II defect was involved in the senescent arrest. The ATP loss was irreversible and accompanied by sustained collapse of mitochondrial membrane potential (Delta psi m), but the ATP loss itself did not seem to be essential in progression to the senescent arrest. The Delta psi m disruption was due to decreased mitochondrial respiration, which was primarily associated with the defective complex II activity. Furthermore, we found that the declined activity of complex II was mainly due to down-regulation of protein expression of the iron-sulfur subunit, which was associated with the irreversibility of the arrest. Finally, we demonstrated that specific inhibition of complex II with 2-thenoyltrifluoroacetone induced overall delay of the cell cycle, suggesting that the delayed arrest by desferroxamine mesylate might be in part due to inhibition of complex II activity. Taken together, our results suggest that complex II might be considered as one of the primary factors to regulate mitochondrial respiratory function by responding to the cellular iron level, thereby influencing cellular growth.

Infrared spectroscopy has been used to examine the oxidized and CO-inhibited forms of Fe-only hydrogenase I from Clostridium pasteurianum. For the oxidized enzyme, five bands are detected in the infrared spectral region between 2100 and 1800 cm(-1). The pattern of infrared bands is consistent with the presence of two terminally coordinated carbon monoxide molecules, two terminally coordinated cyanide molecules, and one bridging carbon monoxide molecule, ligated to the Fe atoms of the active site [2Fe] subcluster. Infrared spectra of the carbon monoxide-inhibited state, prepared using both natural abundance CO and 13CO, indicate that the two terminally coordinated CO ligands that are intrinsic to the enzyme are coordinated to different Fe atoms of the active site [2Fe] subcluster. Irradiation of the CO-inhibited state at cryogenic temperatures gives rise to two species with dramatically different infrared spectra. The first species has an infrared spectrum identical to the spectrum of the oxidized enzyme, and can be assigned as arising from the photolysis of the exogenous CO from the active site. This species, which has been observed in X-ray crystallographic measurements [Lemon, B. J., and Peters, J. W. (2000) J. Am. Chem. Soc. 122, 3793], decays above 150 K. The second light-induced species decays above 80 K and is characterized by loss of the infrared band associated with the Fe bridging CO at 1809 cm(-1). Potential models for the second photolysis event are discussed.

Changes in iron homeostasis have been implicated in cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX). Certain products of DOX metabolism, like the secondary alcohol doxorubicinol (DOXol) or reactive oxygen species (ROS), may contribute to cardiotoxicity by inactivating iron regulatory proteins (IRP) that modulate the fate of mRNAs for transferrin receptor and ferritin. It is important to know whether DOXol and ROS act by independent or combined mechanisms. Therefore, we monitored IRP activities in H9c2 rat embryo cardiomyocytes exposed to DOX or to analogues which were selected to achieve a higher formation of secondary alcohol metabolite (daunorubicin), a concomitant increase of alcohol metabolite and decrease of ROS (5-iminodaunorubicin), or a defective conversion to alcohol metabolite (mitoxantrone). On the basis of such multiple comparisons, we characterized that DOXol was able to remove iron from the catalytic Fe-S cluster of cytoplasmic aconitase, making this enzyme switch to the cluster-free IRP-1. ROS were not involved in this step, but they converted the IRP-1 produced by DOXol into a null protein which did not bind to mRNA, nor was it able to switch back to aconitase. DOX was also shown to inactivate IRP-2, which does not assemble or disassemble a Fe-S cluster. Comparisons between DOX and the analogues revealed that IRP-2 was inactivated only by ROS. Thus, DOX can inactivate both IRP through a sequential action of DOXol and ROS on IRP-1 or an independent action of ROS on IRP-2. This information serves guidelines for designing anthracyclines that spare iron homeostasis and induce less severe cardiotoxicity.

We have investigated the functional relationship between three of the prosthetic groups of Escherichia coli nitrate reductase A (NarGHI): the two hemes of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster of the electron-transfer subunit (NarH). In two site-directed mutants (NarGHI(H56R) and NarGHI(H205Y)) that lack the highest potential heme of NarI (heme b(H)), a large negative DeltaE(m,7) is elicited on the NarH [3Fe-4S] cluster, suggesting a close juxtaposition of these two centers in the holoenzyme. In a mutant retaining heme b(H), but lacking heme b(L) (NarGHI(H66Y)), there is no effect on the NarH [3Fe-4S] cluster redox properties. These results suggest a role for heme b(H) in electron transfer to the [3Fe-4S] cluster. Studies of the pH dependence of the [3Fe-4S] cluster, heme b(H), and heme b(L) E(m) values suggest that significant deprotonation is only observed during oxidation of the latter heme (a pH dependence of -36 mV pH(-1)). In NarI expressed in the absence of NarGH [NarI(DeltaGH)], apparent exposure of heme b(H) to the aqueous milieu results in both it and heme b(L) having E(m) values with pH dependencies of approximately -30 mV pH(-1). These results are consistent with heme b(H) being isolated from the aqueous milieu and pH effects in the holoenzyme. Optical spectroscopy indicates that inhibitors such as HOQNO and stigmatellin bind and inhibit oxidation of heme b(L) but do not inhibit oxidation of heme b(H). Fluorescence quench titrations indicate that HOQNO binds with higher affinity to the reduced form of NarGHI than to the oxidized form. Overall, the data support the following model for electron transfer through the NarI region of NarGHI: Q(P) site --> heme b(L) --> heme b(H) --> [3Fe-4S] cluster.

N,N'-dicyclohexylcarbodiimide (DCCD) has been reported to inhibit proton translocation by cytochrome bc(1) and b(6)f complexes without significantly altering the rate of electron transport, a process referred to as decoupling. To understand the possible role of DCCD in inhibiting the protonogenic reactions of cytochrome bc(1) complex, we investigated the effect of DCCD modification on flash-induced electron transport and electrochromic bandshift of carotenoids in Rb. sphaeroides chromatophores. DCCD has two distinct effects on phase III of the electrochromic bandshift of carotenoids reflecting the electrogenic reactions of the bc(1) complex. At low concentrations, DCCD increases the magnitude of the electrogenic process because of a decrease in the permeability of the membrane, probably through inhibition of F(o)F(1). At higher concentrations (>150 microM), DCCD slows the development of phase III of the electrochromic shift from about 3 ms in control preparations to about 23 ms at 1.2 mM DCCD, without significantly changing the amplitude. DCCD treatment of chromatophores also slows down the kinetics of flash-induced reduction of both cytochromes b and c, from 1.5-2 ms in control preparations to 8-10 ms at 0.8 mM DCCD. Parallel slowing of the reduction of both cytochromes indicates that DCCD treatment modifies the reaction of QH(2) oxidation at the Q(o) site. Despite the similarity in the kinetics of both cytochromes, the onset of cytochrome c re-reduction is delayed 1-2 ms in comparison to cytochrome b reduction, indicating that DCCD inhibits the delivery of electrons from quinol to heme c(1). We conclude that DCCD treatment of chromatophores leads to modification of the rate of Q(o)H(2) oxidation by the iron-sulfur protein (ISP) as well as the donation of electrons from ISP to c(1), and we discuss the results in the context of the movement of ISP between the Q(o) site and cytochrome c(1).

Based on the atomic structures of the mitochondrial cytochrome bc(1) complex, it has been proposed that the soluble domain of the [2Fe-2S] Rieske iron-sulfur protein (ISP) must rotate by ca. 60 degrees and translate through an appreciable distance between two binding sites, proximal to cytochrome c(1) and to the lumen-side quinol binding site. Such motional freedom implies that the electron-transfer rate should be affected by the lumenal viscosity. The flash-induced oxidation of cytochrome f, the chloroplast analogue of cytochrome c(1), was found to be inhibited reversibly by increased lumenal viscosity, as was the subsequent reduction of both cytochrome b(6) and cytochrome f. The rates of these three redox reactions correlated inversely with lumenal viscosity over a viscosity range of 1-10 cP. Reduction of cytochrome b(6) and cytochrome f was not concerted. The rate of cytochrome f reduction was observed to be approximately half that of cytochrome b(6) regardless of the actual viscosity, implying that the path length traversed by the ISP in reduction of cytochrome f is twice that of cytochrome b(6). This suggests that upon initiation of electron transfer by a light flash, cytochrome b(6) reduction requires movement of reduced ISP from an initial position predominantly proximal to cytochrome f, apparently favored by the reduced ISP, to the quinol binding site at which the oxidant-induced reduction of cytochrome b(6) is initiated. Subsequent reduction of cytochrome f requires the additional movement of the ISP back to a site proximal to cytochromef. There is no discernible viscosity dependence for cytochrome b(6) reduction under oxidizing conditions, presumably because the oxidized ISP preferentially binds proximal to the quinone binding niche. The dependence of the cytochrome redox reaction on ambient viscosity implies that the tethered diffusional motion of the ISP is part of the rate limitation for charge transfer through the b(6)f complex.

A site for the binding of exogenously added carbon monoxide has been identified at the active site of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. The binding and inhibition of carbon monoxide have been exploited in biochemical and spectroscopic studies to gain mechanistic insights. In the present study, we have taken advantage of the ability to generate an irreversibly carbon monoxide bound state of CpI. The crystallization and structural characterization of CpI inhibited in the presence of carbon monoxide indicates the addition of a single molecule of carbon monoxide. The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site of carbon monoxide binding at the active site of CpI. Carbon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule in the as crystallized native state of CpI that has been previously suggested to be a potential site of reversible hydrogen oxidation. Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), providing a rational potential mechanism for inhibition of reversible hydrogen oxidation at the active site of CpI.

The interaction of the inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with the Rieske protein of the chloroplast b6f complex has been studied by EPR. All three redox states of DBMIB were found to interact with the iron-sulphur cluster. The presence of the oxidised form of DBMIB altered the equilibrium distribution of the Rieske protein's conformational substates, strongly favouring the proximal position close to heme bL. In addition to this conformational effect, DBMIB shifted the pK-value of the redox-linked proton involved in the iron-sulphur cluster's redox transition by about 1.5 pH units towards more acidic values. The implications of these results with respect to the interaction of the native quinone substrate and the Rieske cluster in cytochrome bc complexes are discussed.