Metal–metal bonds in biology Lindahl, Paul A.
Journal of inorganic biochemistry,
01/2012, Letnik:
106, Številka:
1
Journal Article
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Nickel-containing carbon monoxide dehydrogenases, acetyl-CoA synthases, nickel–iron hydrogenases, and diron hydrogenases are distinct metalloenzymes yet they share a number of important ...characteristics. All are O
2-sensitive, with active-sites composed of iron and/or nickel ions coordinated primarily by sulfur ligands. In each case, two metals are juxtaposed at the “heart” of the active site, within range of forming metal–metal bonds. These active-site clusters exhibit multielectron redox abilities and must be reductively activated for catalysis. Reduction potentials are milder than expected based on formal oxidation state changes. When reductively activated, each cluster attacks an electrophilic substrate via an oxidative addition reaction. This affords a two-electron-reduced substrate bound to one or both metals of an oxidized cluster. M–M bonds have been established in hydrogenases where they serve to initiate the oxidative addition of protons and perhaps stabilize active sites in multiple redox states. The same may be true of the CODH and ACS active sites—Ni–Fe and Ni–Ni bonds in these sites may play critical roles in catalysis, stabilizing low-valence states and initiating oxidative addition of CO
2 and methyl group cations, respectively. In this article, the structural and functional commonalities of these metalloenzyme active sites are described, and the case is made for the formation and use of metal–metal bonds in each enzyme mentioned. As a post-script, the importance of Fe–Fe bonds in the nitrogenase FeMoco active site is discussed.
The possibility that metal–metal bonds form in metalloenzymes and are used in catalysis is highlighted using iron–iron hydrogenase, nickel–iron hydrogenase, nickel-containing carbon monoxide dehydrogenase, acetyl-coenzyme A synthase and nitrogenase as examples. These bonds are proposed to form in reduced states and function to initiate oxidative addition reactions. If verified, a new motif in mechanistic bioinorganic chemistry would be established.
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The HFE (Homeostatic Fe regulator) gene is commonly mutated in hereditary hemochromatosis. Blood of (HFE)(−/−) mice and of humans with hemochromatosis contains toxic nontransferrin-bound iron (NTBI) ...which accumulates in organs. However, the chemical composition of NTBI is uncertain. To investigate, HFE(−/−) mice were fed iron-deficient diets supplemented with increasing amounts of iron, with the expectation that NTBI levels would increase. Blood plasma was filtered to obtain retentate and flow-through solution fractions. Liquid chromatography detected by inductively coupled plasma mass spectrometry of flow-through solutions exhibited low-molecular-mass iron peaks that did not increase intensity with increasing dietary iron. Retentates yielded peaks due to transferrin (TFN) and ferritin, but much iron in these samples adsorbed onto the column. Retentates treated with the chelator deferoxamine (DFO) yielded a peak that comigrated with the Fe–DFO complex and originated from iron that adhered to the column in the absence of DFO. Additionally, plasma from younger and older 57Fe-enriched HFE mice were separately pooled and concentrated by ultrafiltration. After removing contributions from contaminating blood and TFN, Mössbauer spectra were dominated by features due to magnetically interacting FeIII aggregates, with greater intensity in the spectrum from the older mice. Similar features were generated by adding 57FeIII to “pseudo plasma”. Aggregation was unaffected by albumin or citrate at physiological concentrations, but DFO or high citrate concentrations converted aggregated FeIII into high-spin FeIII complexes. FeIII aggregates were retained by the cutoff membrane and adhered to the column, similar to the behavior of NTBI. A model is proposed in which FeII entering blood is oxidized, and if apo-TFN is unavailable, the resulting FeIII ions coalesce into FeIII aggregates, a.k.a. NTBI.
Iron is an essential transition metal for all eukaryotic cells, and its trafficking throughout the cell is highly regulated. However, the overall cellular mechanism of regulation is poorly understood ...despite knowing many of the molecular players involved. Here, an ordinary-differential-equations (ODE) based kinetic model of iron trafficking within a growing yeast cell was developed that included autoregulation. The 9-reaction 8-component in-silico cell model was solved under both steady-state and time-dependent dynamical conditions. The ODE for each component included a dilution term due to cell growth. Conserved rate relationships were obtained from the null space of the stoichiometric matrix, and the reduced-row-echelon-form was used to distinguish independent from dependent rates. Independent rates were determined from experimentally estimated component concentrations, cell growth rates, and the literature. Simple rate-law expressions were assumed, allowing rate-constants for each reaction to be estimated. Continuous Heaviside logistical functions were used to regulate rate-constants. These functions acted like valves, opening or closing depending on component "sensor" concentrations. Two cellular regulatory mechanisms were selected from 134,217,728 possibilities using a novel approach involving 6 mathematically-defined filters. Three cellular states were analyzed including healthy wild-type cells, iron-deficient wild-type cells, and a frataxin-deficient strain of cells characterizing the disease Friedreich's Ataxia. The model was stable toward limited perturbations, as determined by the eigenvalues of Jacobian matrices. Autoregulation allowed healthy cells to transition to the diseased state when triggered by a mutation in frataxin, and to the iron-deficient state when cells are placed in iron-deficient growth medium. The in-silico phenotypes observed during these transitions were similar to those observed experimentally. The model also predicted the observed effects of hypoxia on the diseased condition. A similar approach could be used to solve ODE-based kinetic models associated with other biochemical processes operating within growing cells.
Copper is essential for all eukaryotic cells but many details of how it is trafficked within the cell and how it is homeostatically regulated remain uncertain. Here, we characterized the copper ...content of cytosol and mitochondria using liquid chromatography with ICP-MS detection. Chromatograms of cytosol exhibited over two dozen peaks due to copper proteins and coordination complexes. Yeast cells respiring on minimal media did not regulate copper import as media copper concentration increased; rather, they imported copper at increasing rates while simultaneously increasing the expression of metallothionein CUP1 which then sequestered most of the excessive imported copper. Peak intensities due to superoxide dismutase SOD1, other copper proteins, and numerous coordination complexes also increased, but not as drastically. The labile copper pool was unexpectedly diverse and divided into two groups. One group approximately comigrated with copper-glutathione, -cysteine, and -histidine standards; the other developed only at high media copper concentrations and at greater elution volumes. Most cytosolic copper arose from copper-bound proteins, especially CUP1. Cytosol contained an unexpectedly high percentage of apo-copper proteins and apo-coordination complexes. Copper-bound forms of non-CUP1 proteins and complexes coexisted with apo-CUP1 and with the chelator BCS. Both experiments suggest unexpectedly stable-binding copper proteins and coordination complexes in cytosol. COX17Δ cytosol chromatograms were like those of WT cells. Chromatograms of soluble mitochondrial extracts were obtained, and mitoplasting helped distinguish copper species in the intermembrane space versus in the matrix/inner membrane. Issues involving the yeast copperome, copper homeostasis, labile copper pool, and copper trafficking are discussed.
Iron-sulfur cluster (ISC) assembly occurs in both mitochondria and cytosol. Mitochondria are thought to export a low-molecular-mass (LMM) iron and/or sulfur species which is used as a substrate for ...cytosolic ISC assembly. This species, called X-S or (Fe-S)
, has not been directly detected. Here, an assay was developed in which mitochondria were isolated from
Fe-enriched cells and incubated in various buffers. Thereafter, mitochondria were separated from the supernatant, and both fractions were investigated by ICP-MS-detected size exclusion liquid chromatography. Aqueous
Fe
in the buffer declined upon exposure to intact
Fe-enriched mitochondria. Some
Fe was probably surface-absorbed but some was incorporated into mitochondrial iron-containing proteins when mitochondria were activated for ISC biosynthesis. When activated, mitochondria exported/released two LMM nonproteinaceous iron complexes. One species, which comigrated with an Fe-ATP complex, developed faster than the other Fe species, which also comigrated with phosphorus. Both were enriched in
Fe and
Fe, suggesting that the added
Fe entered a pre-existing pool of
Fe, which was also the source of the exported species. When
Fe-loaded
Fe-enriched mitochondria were mixed with isolated cytosol and activated, multiple cytosolic proteins became enriched with Fe. No incorporation was observed when
Fe was added directly to the cytosol in the absence of mitochondria. This suggests that a different Fe source in mitochondria, the one enriched mainly with
Fe, was used to export a species that was ultimately incorporated into cytosolic proteins. Iron from buffer was imported into mitochondria fastest, followed by mitochondrial ISC assembly, LMM iron export, and cytosolic ISC assembly.
Liquid chromatography, mass spectrometry, and metal analyses of cytosol and mitochondrial filtrates from healthy copper-replete Saccharomyces cerevisiae cells revealed that metallothionein CUP1 was a ...notable copper-containing species in both compartments, with its abundance dependent upon the level of copper supplementation in the growth media. Electrospray ionization mass spectrometry of cytosol and soluble mitochondrial filtrates displayed a full isotopologue pattern of CUP1 in which the first eight amino acid residues were truncated and eight copper ions were bound. Neither apo-CUP1 nor intermediate copper-bound forms were detected, but chelator treatment could generate apo-CUP1. Mitoplasting revealed that mitochondrial CUP1 was located in the intermembrane space. Fluorescence microscopy demonstrated that 34 kDa CUP1-GFP entered the organelle, discounting the possibility that 7 kDa CUP1 enters folded and metalated through outer membrane pores. How CUP1 enters mitochondria remains unclear, as does its role within the organelle. Although speculative, mitochondrial CUP1 may limit the concentrations of low-molecular-mass copper complexes in the organelle.
Abstract
One hundred proteins in Saccharomyces cerevisiae are known to contain iron. These proteins are found mainly in mitochondria, cytosol, nuclei, endoplasmic reticula, and vacuoles. Cells also ...contain non-proteinaceous low-molecular-mass labile iron pools (LFePs). How each molecular iron species interacts on the cellular or systems’ level is underdeveloped as doing so would require considering the entire iron content of the cell—the ironome. In this paper, Mössbauer (MB) spectroscopy was used to probe the ironome of yeast. MB spectra of whole cells and isolated organelles were predicted by summing the spectral contribution of each iron-containing species in the cell. Simulations required input from published proteomics and microscopy data, as well as from previous spectroscopic and redox characterization of individual iron-containing proteins. Composite simulations were compared to experimentally determined spectra. Simulated MB spectra of non-proteinaceous iron pools in the cell were assumed to account for major differences between simulated and experimental spectra of whole cells and isolated mitochondria and vacuoles. Nuclei were predicted to contain ∼30 μM iron, mostly in the form of Fe4S4 clusters. This was experimentally confirmed by isolating nuclei from 57Fe-enriched cells and obtaining the first MB spectra of the organelle. This study provides the first semi-quantitative estimate of all concentrations of iron-containing proteins and non-proteinaceous species in yeast, as well as a novel approach to spectroscopically characterizing LFePs.
Graphical Abstract
Graphical Abstract
Lindahl and Vali consider all iron-containing proteins in a yeast cell in simulating the expected Mössbauer spectrum of whole yeast cells.
Objectives This study assessed pharmacodynamic (PD) response to the reduced prasugrel maintenance dose of 5 mg in very elderly (VE) patients (≥75 years of age). Background In the TRITON–TIMI 38 ...(TRial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet InhibitioN with Prasugrel–Thrombolysis In Myocardial Infarction 38) study prasugrel 10 mg reduced ischemic events versus clopidogrel 75 mg, but increased bleeding in VE patients. Methods We examined PD and active metabolite pharmacokinetics (PKs) with prasugrel 5 and 10 mg and clopidogrel 75 mg in a 3-period (12 days each) blinded, crossover study in VE (n = 73; mean: 79 ± 3 years of age) or (n = 82) nonelderly (NE) (≥45 to <65 years of age; mean: 56 ± 5 years of age) stable coronary artery disease (CAD) patients receiving background aspirin. Assays included light transmission aggregometry (LTA), VerifyNow P2Y12 (VN-P2Y12), and vasodilator-associated stimulated phosphoprotein (VASP). The primary comparison was noninferiority of maximum platelet aggregation (MPA) comparing the median for prasugrel 5 mg in VE versus the 75th percentile for prasugrel 10 mg in NE, using a pre-specified 1-sided 97.5% confidence interval for the difference <15%. Results Prasugrel 5 mg in VE met the primary PD noninferiority criterion versus prasugrel 10 mg in NE. For prasugrel 5 mg, MPA was significantly lower (57 ± 14%) than clopidogrel (63 ± 14%; p < 0.001) in VE but higher than prasugrel 10 mg in NE (46 ± 12%; p < 0.001). PD response by LTA, VN-P2Y12, and VASP during all treatments appeared similar between age cohorts. Prasugrel 5 mg resulted in fewer VE poor responders than clopidogrel. Rates of mild bleeding were higher with prasugrel 10 mg but similar for prasugrel 5 mg versus clopidogrel 75 mg. Conclusions In aspirin-treated stable CAD patients, prasugrel 5 mg in VE attenuated platelet inhibition while meeting pre-specified noninferiority criterion versus prasugrel 10 mg in NE, with significantly better PD response and fewer poor responders compared to clopidogrel 75 mg in VE. (Comparison of Prasugrel and Clopidogrel in Very Elderly and Non-Elderly Patients With Stable Coronary Artery Disease GENERATIONS; NCT01107912 )
Labile low-molecular-mass (LMM) transition metal complexes play essential roles in metal ion trafficking, regulation, and signalling in biological systems, yet their chemical identities remain ...largely unknown due to their rapid ligand-exchange rates and weak M–L bonds. Here, an
Escherichia coli
cytosol isolation procedure was developed that was devoid of detergents, strongly coordinating buffers, and EDTA. The interaction of the metal ions from these complexes with a SEC column was minimized by pre-loading the column with
67
ZnSO
4
and then monitoring
66
Zn and other metals by inductively coupled plasma mass spectrometry (ICP-MS) when investigating cytosolic ultrafiltration flow-through-solutions (FTSs). Endogenous cytosolic salts suppressed ESI-MS signals, making the detection of metal complexes difficult. FTSs contained ca. 80 µM Fe, 15 µM Ni, 13 µM Zn, 10 µM Cu, and 1.4 µM Mn (after correcting for dilution during cytosol isolation). FTSs exhibited 2–5 Fe, at least 2 Ni, 2–5 Zn, 2–4 Cu, and at least 2 Mn species with apparent masses between 300 and 5000 Da. Fe(ATP), Fe(GSH), and Zn(GSH) standards were passed through the column to assess their presence in FTS. Major LMM sulfur- and phosphorus-containing species were identified. These included reduced and oxidized glutathione, methionine, cysteine, orthophosphate, and common mono- and di-nucleotides such as ATP, ADP, AMP, and NADH. FTSs from cells grown in media supplemented with one of these metal salts exhibited increased peak intensity for the supplemented metal indicating that the size of the labile metal pools in
E. coli
is sensitive to the concentration of nutrient metals.
Nickel serves critical roles in the metabolism of E. coli and many prokaryotes. Many details of nickel trafficking are unestablished, but a nonproteinaceous low-molecular-mass (LMM) labile nickel ...pool (LNiP) is thought to be involved. The portion of the cell lysate that flowed through a 3 kDa cutoff membrane, which ought to contain this pool, was analyzed by size-exclusion and hydrophilic interaction chromatographies (SEC and HILIC) with detection by inductively coupled plasma (ICP) and electrospray ionization (ESI) mass spectrometries. Flow-through-solutions (FTSs) contained 11–15 μM Ni, which represented most Ni in the cell. Chromatograms exhibited 4 major Ni-detected peaks. MS analysis of FTS and prepared nickel complex standards established that these peaks arose from Ni(II) coordinated to oxidized glutathione, histidine, aspartate, and ATP. Surprisingly, Ni complexes with reduced glutathione or citrate were not members of the LNiP under the conditions examined. Aqueous Ni(II) ions were absent in the FTS. Detected complexes were stable in chelator-free buffer but were disrupted by treatment with 1,10-phenanthroline or citrate. Titrating FTS with additional NiSO4 suggested that the total nickel-binding capacity of cytosol is approximately 20–45 μM. Members of the LNiP are probably in rapid equilibrium. Previously reported binding constants to various metalloregulators may have overestimated the relevant binding strength in the cell because aqueous metal salts were used in those determinations. The LNiP may serve as both a Ni reservoir and buffer, allowing cells to accommodate a range of Ni concentrations. The composition of the LNiP may change with cellular metabolism and nutrient status.
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