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1.
Biochemistry ; 52(35): 6030-9, 2013 Sep 03.
Article in English | MEDLINE | ID: mdl-23899293

ABSTRACT

The maturation of [NiFe]-hydrogenase in Escherichia coli is a complex process involving many steps and multiple accessory proteins. The two accessory proteins HypA and HypB interact with each other and are thought to cooperate to insert nickel into the active site of the hydrogenase-3 precursor protein. Both of these accessory proteins bind metal individually, but little is known about the metal-binding activities of the proteins once they assemble together into a functional complex. In this study, we investigate how complex formation modulates metal binding to the E. coli proteins HypA and HypB. This work lead to a re-evaluation of the HypA nickel affinity, revealing a KD on the order of 10(-8) M. HypA can efficiently remove nickel, but not zinc, from the metal-binding site in the GTPase domain of HypB, a process that is less efficient when complex formation between HypA and HypB is disrupted. Furthermore, nickel release from HypB to HypA is specifically accelerated when HypB is loaded with GDP, but not GTP. These results are consistent with the HypA-HypB complex serving as a transfer step in the relay of nickel from membrane transporter to its final destination in the hydrogenase active site and suggest that this complex contributes to the metal fidelity of this pathway.


Subject(s)
Carrier Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , GTP-Binding Proteins/metabolism , Hydrogenase/metabolism , Metals/metabolism , Base Sequence , DNA Primers , Intracellular Signaling Peptides and Proteins , Spectrometry, Mass, Electrospray Ionization
2.
J Biol Inorg Chem ; 16(6): 857-68, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21544686

ABSTRACT

Biosynthesis of the metallocenter in the active site of the [NiFe] hydrogenase enzyme requires the accessory protein HypB, which is a metal-binding GTPase. In this study, the interplay between the individual activities of Escherichia coli HypB was examined. The full-length protein undergoes nucleotide-responsive dimerization that is disrupted upon mutation of L242 and L246 to alanine. This mutant HypB is monomeric under all of the conditions investigated but the inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric protein has metal-binding behavior similar to that of wild-type HypB. Furthermore, expression of L242A/L246A HypB in vivo results in hydrogenase activity that is approximately half of the activity produced by the wild-type control, suggesting that dimerization of HypB does not have a critical role in the hydrogenase maturation pathway. In contrast, the GTPase activity of HypB is modulated by metal loading of the protein. These results provide insight into the role of HypB in hydrogenase biosynthesis.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Animals , Escherichia coli Proteins/genetics , GTP Phosphohydrolases/genetics , GTP-Binding Proteins/genetics , Guanosine Diphosphate/metabolism , Metals/chemistry , Protein Binding , Protein Conformation , Protein Multimerization
3.
Biochem Biophys Res Commun ; 401(1): 69-74, 2010 Oct 08.
Article in English | MEDLINE | ID: mdl-20833145

ABSTRACT

As³+ bound to the two-domain, recombinant human metallothionein (isoform 1a) is stable at pH 7 and translocates via protein-protein interactions to other metallothionein proteins. The data show As³+ transfer from the two-domain ß-α-hMT to binding sites in the isolated apo-ß-hMT and apo-α-hMT. Under conditions of equilibrium, apo- and partially-metallated species coexist indicating that noncooperative demetallation of the As(6)-ßα-hMT occurrs. As³+ transfer under conditions (pH 7) where the free As³+ ion is not stable, provides evidence that Cd²+ and Zn²+ transfer may also take place through protein-protein interactions and that partially metallated Cd-MT and Zn-MT would be stable.


Subject(s)
Arsenic/metabolism , Metallothionein/metabolism , Cadmium/metabolism , Enzyme Stability , Humans , Hydrogen-Ion Concentration , Ion Transport , Metallothionein/chemistry , Metallothionein/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Zinc/metabolism
4.
Biochem Biophys Res Commun ; 396(2): 206-12, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20398631

ABSTRACT

Bismuth compounds are currently used to treat gastric ailments and to prevent the toxic side effects of cancer treatments. The affinity of bismuth for binding to sulfur compounds has been reported and one such target biomolecule is the cysteine-rich metalloprotein metallothionein. Renal mammalian metallothionein has been shown to be induced by Bi salts, with the Bi(3+) binding to the renal MT. However, the exact metal-to-metallothionein stoichiometric ratios for the 2-domain betaalpha mammalian protein and the individual beta and alpha domain fragments remain unknown. We now report that the maximum metal-to-MT stoichiometries for the individual domain fragments and the entire 2-domain protein are Bi(3)-S(9)-betahMT, Bi(4)-S(11)-alphahMT, and Bi(7)-S(20)-betaalphahMT, respectively. Electrospray mass spectrometry data also unambiguously show the existence of partially metalated Bi-containing MT species during the titration of apo-MT with Bi(3+), which demonstrates that Bi-metalation to MT occurs in a noncooperative manner.


Subject(s)
Antacids/metabolism , Bismuth/metabolism , Cysteine/metabolism , Metallothionein/metabolism , Antacids/chemistry , Binding Sites , Bismuth/chemistry , Cysteine/chemistry , Humans , Metallothionein/chemistry , Protein Binding , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization
5.
J Inorg Biochem ; 104(3): 232-44, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20079542

ABSTRACT

Metallothionein (MT) is a prominent metal-binding protein and in mammalian systems contains a two-domain betaalpha motif, while in lower life forms MT often consists of only a single-domain structure. There are also unusual MTs from American oysters that consist of multiple domains (from one to four alpha domains). This report details the study of the As(3+)-metalation to two different concatenated triple beta and alpha domain MTs using time-resolved electrospray ionization mass spectrometry (ESI MS). Analysis of kinetic ESI MS data show that alphaalphaalpha human MT and betabetabeta human MT bind As(3+) in a noncooperative manner and involves up to 11 sequential bimolecular reactions. We report the complete progress of the reactions for the As(3+)-metalation of both triple-domain MTs from zero and up to 9 (betabetabeta) or 10As(3+) ions (alphaalphaalpha). The rate constants for the As(3+)-metalation are reported for both the betabetabeta and alphaalphaalpha human MT. At room temperature (298K) and pH3.5, the sequential individual rate constants, k(n) (n=1-9) for the As(3+)-metalation of betabetabetahMT starting at k(1betabetabeta) are 40, 36, 37, 26, 27, 17, 12, 6, and 1M(-1)s(-1); while at room temperature (298K) and pH3.5, the sequential individual rate constants, k(n) (n=1-10) for the As(3+)-metalation of alphaalphaalphahMT starting at k(1alphaalphaalpha) are 52, 45, 46, 42, 38, 36, 29, 25, 14, and 6M(-1)s(-1). The trend in the rate constant values reported for these two triple-domain MT proteins supports our previous proposal that the rate constant values are proportionally related to the total number of equivalent binding sites. The rate of binding for the 1st As(3+) is the fastest we have determined for any MT to date. Additionally, we propose that the data show for the first time for any MT species, that interdomain metalation occurs in the binding of the 10th and 11th As(3+) to alphaalphaalphahMT.


Subject(s)
Arsenic/metabolism , Metallothionein/chemistry , Metallothionein/metabolism , Protein Conformation , Amino Acid Sequence , Animals , Arsenic/chemistry , Binding Sites , Biological Evolution , Humans , Metallothionein/genetics , Molecular Sequence Data , Protein Binding , Spectrometry, Mass, Electrospray Ionization
6.
Biochemistry ; 48(37): 8806-16, 2009 Sep 22.
Article in English | MEDLINE | ID: mdl-19655782

ABSTRACT

The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.


Subject(s)
Algal Proteins/chemistry , Arsenic/chemistry , Arsenic/metabolism , Fucus/chemistry , Metallothionein/chemistry , Metallothionein/metabolism , Algal Proteins/metabolism , Amino Acid Sequence , Arsenic/toxicity , Fucus/drug effects , Fucus/metabolism , Kinetics , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization
7.
Dalton Trans ; (28): 5425-33, 2009 Jul 28.
Article in English | MEDLINE | ID: mdl-19587982

ABSTRACT

Metallothionein are small, cysteine-rich, metal-binding proteins that are found ubiquitously in nature. Most metallothioneins bind multiple metals in two well-defined metal-thiolate clusters. This perspective discusses the use of optical spectroscopy to study the metalation of metallothioneins and the emergence of electrospray ionization mass spectrometry as a means of studying the mechanism of metalation for metallothioneins. A brief history of past kinetic studies of cadmium metallothioneins and recent kinetic study advances for the arsenic metalation of metallothionein will be presented. Lastly, a possible functional role for the two-domain structure of metallothionein will be briefly discussed.


Subject(s)
Metallothionein/metabolism , Metals/metabolism , Kinetics , Magnetic Resonance Spectroscopy , Metallothionein/chemistry , Models, Molecular , Protein Binding , Spectrometry, Mass, Electrospray Ionization
8.
IUBMB Life ; 61(4): 438-46, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19319961

ABSTRACT

Metalloproteins represent approximately 30% of all proteins known, yet our understanding of the structures of these metalloproteins, the metal content, and the mechanism for metalation are still very limited. One of the most studied metalloproteins is the ubiquitous metallothionein (MT), which in mammals contains two metal-binding domains: a 9-cysteine beta domain and a 11-cysteine alpha domain. Metals are coordinated in MT via the cysteinyl thiols present in the primary amino acid sequence and the geometry is controlled by the metal ion. This short review discusses the use of optical spectroscopy to study the metalation of MT with particular emphasis on the benefits and pitfalls involved. Further, the new properties of MT that have been revealed using electrospray ionization mass spectrometry in recent metalation studies will also be discussed.


Subject(s)
Metallothionein/chemistry , Metallothionein/metabolism , Metals/metabolism , Models, Molecular , Protein Conformation , Protein Structure, Tertiary , Kinetics , Mass Spectrometry
9.
J Am Chem Soc ; 130(50): 17016-28, 2008 Dec 17.
Article in English | MEDLINE | ID: mdl-19053406

ABSTRACT

Metallothionein (MT) is ubiquitous in Nature, underlying MT's importance in the cellular chemistry of metals. Mammalian MT consists of two metal-binding domains while microorganisms like cyanobacteria consist of a single metal-binding domain MT. The evolution of a two-domain protein has been speculated on for some time; however, no conclusive evidence explaining the evolutionary necessity of the two-domain structure has been reported. The results presented in this report provide the complete kinetic analysis and subsequent mechanism of the As(3+)-metalation of the two-domain beta alpha hMT and the isolated single domain fragments using time- and temperature-resolved electrospray ionization mass spectrometry. The mechanism for beta alpha hMT binding As(3+) is noncooperative and involves six sequential bimolecular reactions in which the alpha domain binds As(3+) first followed by the beta domain. At room temperature (295 K) and pH 3.5, the sequential individual rate constants, k(n) (n = 1-6) for the As(3+)-metalation of beta alpha hMT starting at k(1beta alpha) are 25, 24, 19, 14, 8.7, and 3.7 M(-1)s(-1). The six rate constants follow an almost linear trend directly dependent on the number of unoccupied sites for the incoming metal. Analysis of the temperature-dependent kinetic electrospray ionization mass spectra data allowed determination of the activation energy for the formation of As(1)-H(17)-beta alpha hMT (14 kJ mol(-1)) and As(2-6)-beta alpha hMT (22 kJ mol(-1)). On the basis of the increased rate of metalation for the two-domain protein when compared with the isolated single-domain, we propose that there is an evolutionary advantage for the two-domain MT structures in higher organism, which allows MT to bind metals faster and, therefore, be a more efficient metal scavenger.


Subject(s)
Arsenic/chemistry , Metallothionein/chemistry , Arsenic/metabolism , Humans , Kinetics , Metallothionein/metabolism , Models, Molecular , Protein Binding , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Temperature
10.
Exp Biol Med (Maywood) ; 231(9): 1488-99, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17018871

ABSTRACT

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.


Subject(s)
Metallothionein/metabolism , Peptides/metabolism , Binding Sites , Metallothionein/chemistry , Protein Conformation , Protein Folding
11.
Biochem Biophys Res Commun ; 351(1): 229-33, 2006 Dec 08.
Article in English | MEDLINE | ID: mdl-17054910

ABSTRACT

The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd(2+) ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd(3)Cys(9) and Cd(4)Cys(11) based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.


Subject(s)
Cadmium/chemistry , Metallothionein/chemistry , Oligochaeta/metabolism , Animals , Binding Sites , Circular Dichroism , Protein Binding , Protein Interaction Mapping , Spectrometry, Mass, Electrospray Ionization
12.
J Am Chem Soc ; 128(38): 12473-83, 2006 Sep 27.
Article in English | MEDLINE | ID: mdl-16984198

ABSTRACT

The number of reported cases of chronic arsenic poisoning is on the rise throughout the world, making the study of the long-term effects of arsenic critical. As(3+) binds readily to biological thiols, including mammalian metallothionein (MT), which is an ubiquitous sulfur-rich metalloprotein known to coordinate a wide range of metals. The two-domain mammalian protein binds divalent metals (M) into two metal-thiolate clusters with stoichiometries of M(3)S(cys9) (beta) and M(4)S(cys11) (alpha). We report that As(3+) binds with stoichiometries of As(3)S(cys9) (beta) and As(3)S(cys11) (alpha) to the recombinant human metallothionein (rhMT) isoform 1a protein. Further, we report the complete kinetic analysis of the saturation reactions of the separate alpha and beta domains of rhMT with As(3+). Speciation in the metalation reactions was determined using time- and temperature-resolved electrospray ionization mass spectrometry. The binding reaction of As(3+) to the alpha and beta MT domains is shown to be noncooperative and involves three sequential, bimolecular metalation steps. The analyses allow for the first time the complete simulation of the experimental data for the stepwise metalation reaction of MT showing the relative concentrations of the metal-free, apo MT and each of the As-MT intermediate species as a function of time and temperature. At room temperature (298 K) and pH 3.5, the individual rate constants for the first, second, and third As(3+) binding to apo-alphaMT are 5.5, 6.3, and 3.9 M(-)(1) s(-)(1) and for apo-betaMT the constants are 3.6, 2.0, and 0.6 M(-)(1) s(-)(1). The activation energy for formation of As(1)-H(6)-betaMT is 32 kJ mol(-)(1), for As(2)-H(3)-betaMT it is 35 kJ mol(-)(1), for As(3)-betaMT it is 29 kJ mol(-)(1), for As(1)-H(8)-alphaMT it is 33 kJ mol(-)(1), for As(2)-H(5)-alphaMT it is 29 kJ mol(-)(1), and for As(3)-H(2)-alphaMT it is 23 kJ mol(-)(1).


Subject(s)
Arsenic/chemistry , Arsenic/metabolism , Metallothionein/chemistry , Metallothionein/metabolism , Amino Acid Sequence , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Spectrometry, Mass, Electrospray Ionization , Thermodynamics
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