Metalloprotein is a generic term for aprotein that contains a metal ioncofactor.[1][2] A large proportion of all proteins are part of this category. For instance, at least 1000 human proteins (out of ~20,000) contain zinc-bindingprotein domains[3] although there may be up to 3000 human zinc metalloproteins.[4]
It is estimated that approximately half of allproteins contain ametal.[5] In another estimate, about one quarter to one third of all proteins are proposed to require metals to carry out their functions.[6] Thus, metalloproteins have many different functions incells, such as storage and transport of proteins,enzymes andsignal transduction proteins, or infectious diseases.[7]
Most metals in thehumanbody are bound to proteins. For instance, the relatively high concentration of iron in the human body is mostly due to the iron inhemoglobin.
| Liver | Kidney | Lung | Heart | Brain | Muscle | |
|---|---|---|---|---|---|---|
| Mn (manganese) | 138 | 79 | 29 | 27 | 22 | <4-40 |
| Fe (iron) | 16,769 | 7,168 | 24,967 | 5,530 | 4,100 | 3,500 |
| Co (cobalt) | <2-13 | <2 | <2-8 | --- | <2 | 150 (?) |
| Ni (nickel) | <5 | <5-12 | <5 | <5 | <5 | <15 |
| Cu (copper) | 882 | 379 | 220 | 350 | 401 | 85-305 |
| Zn (zinc) | 5,543 | 5,018 | 1,470 | 2,772 | 915 | 4,688 |
In metalloproteins, metal ions are usually coordinated bynitrogen,oxygen orsulfur centers belonging toamino acid residues of the protein. These donor groups are often provided by side-chains on the amino acid residues. Especially important are theimidazole substituent inhistidine residues,thiolate substituents incysteine residues, andcarboxylate groups provided byaspartate. Given the diversity of the metalloproteome, virtually all amino acid residues have been shown to bind metal centers. The peptide backbone also provides donor groups; these include deprotonatedamides and the amidecarbonyl oxygen centers. Lead(II) binding in natural and artificial proteins has been reviewed.[9]
In addition to donor groups that are provided by amino acid residues, many organiccofactors function asligands. Perhaps most famous are the tetradentate N4macrocyclic ligands incorporated into theheme protein. Inorganic ligands such as sulfide and oxide are also common.
These are the second stage product of protein hydrolysis obtained by treatment with slightly stronger acids and alkalies.
Hemoglobin, which is the principal oxygen-carrier in humans, has four subunits in which theiron(II) ion is coordinated by the planarmacrocyclicligandprotoporphyrin IX (PIX) and theimidazole nitrogen atom of ahistidine residue. The sixth coordination site contains awater molecule or adioxygen molecule. By contrast the proteinmyoglobin, found inmuscle cells, has only one such unit. The active site is located in ahydrophobic pocket. This is important as without it the iron(II) would be irreversiblyoxidized to iron(III). Theequilibrium constant for the formation of HbO2 is such that oxygen is taken up or released depending on thepartial pressure of oxygen in thelungs or in muscle. In hemoglobin the four subunits show a cooperativity effect that allows for easy oxygen transfer from hemoglobin to myoglobin.[10]
In bothhemoglobin andmyoglobin it is sometimes incorrectly stated that the oxygenated species contains iron(III). It is now known that thediamagnetic nature of these species is because the iron(II) atom is in thelow-spin state. Inoxyhemoglobin the iron atom is located in the plane of the porphyrin ring, but in theparamagneticdeoxyhemoglobin the iron atom lies above the plane of the ring.[10] This change in spin state is a cooperative effect due to the highercrystal field splitting and smallerionic radius of Fe2+ in the oxyhemoglobin moiety.
Hemerythrin is another iron-containing oxygen carrier. The oxygen binding site is a binuclear iron center. The iron atoms are coordinated to the protein through thecarboxylate side chains of aglutamate andaspartate and fivehistidine residues. The uptake of O2 by hemerythrin is accompanied by two-electron oxidation of the reduced binuclear center to produce boundperoxide (OOH−). The mechanism of oxygen uptake and release have been worked out in detail.[11][12]
Hemocyanins carry oxygen in the blood of mostmollusks, and somearthropods such as thehorseshoe crab. They are second only to hemoglobin in biological popularity of use in oxygen transport. On oxygenation the twocopper(I) atoms at the active site are oxidized to copper(II) and the dioxygen molecules are reduced to peroxide,O2−
2.[13][14]
Chlorocruorin (as the larger carriererythrocruorin) is an oxygen-binding hemeprotein present in theblood plasma of manyannelids, particularly certain marinepolychaetes.
Oxidation andreduction reactions are not common inorganic chemistry as few organic molecules can act asoxidizing orreducing agents.Iron(II), on the other hand, can easily be oxidized to iron(III). This functionality is used incytochromes, which function aselectron-transfer vectors. The presence of the metal ion allowsmetalloenzymes to perform functions such asredox reactions that cannot easily be performed by the limited set offunctional groups found inamino acids.[15] The iron atom in most cytochromes is contained in aheme group. The differences between those cytochromes lies in the different side-chains. For instance cytochrome a has aheme a prosthetic group and cytochrome b has aheme b prosthetic group. These differences result in different Fe2+/Fe3+redox potentials such that various cytochromes are involved in themitochondrial electron transport chain.[16]
Cytochrome P450 enzymes perform the function of inserting an oxygen atom into a C−H bond, an oxidation reaction.[17][18]
Rubredoxin is an electron-carrier found insulfur-metabolizingbacteria andarchaea. The active site contains an iron ion coordinated by the sulfur atoms of fourcysteine residues forming an almost regulartetrahedron. Rubredoxins perform one-electron transfer processes. Theoxidation state of the iron atom changes between the +2 and +3 states. In both oxidation states the metal ishigh spin, which helps to minimize structural changes.
Plastocyanin is one of the family of bluecopper proteins that are involved inelectron transfer reactions. Thecopper-binding site is described as distortedtrigonal pyramidal.[19] The trigonal plane of the pyramidal base is composed of two nitrogen atoms (N1 and N2) from separate histidines and a sulfur (S1) from a cysteine. Sulfur (S2) from an axial methionine forms the apex. The distortion occurs in the bond lengths between the copper and sulfur ligands. The Cu−S1 contact is shorter (207 pm) than Cu−S2 (282 pm).The elongated Cu−S2 bonding destabilizes the Cu(II) form and increases theredox potential of the protein. The blue color (597 nm peak absorption) is due to the Cu−S1 bond where S(pπ) to Cu(dx2−y2) charge transfer occurs.[20]
In the reduced form of plastocyanin,His-87 will become protonated with apKa of 4.4.Protonation prevents it acting as aligand and the copper site geometry becomestrigonal planar.
Iron is stored as iron(III) inferritin. The exact nature of the binding site has not yet been determined. The iron appears to be present as ahydrolysis product such as FeO(OH). Iron is transported bytransferrin whose binding site consists of twotyrosines, oneaspartic acid and onehistidine.[21] The human body has no controlled mechanism for excretion of iron.[22] This can lead toiron overload problems in patients treated withblood transfusions, as, for instance, with β-thalassemia. Iron is actually excreted in urine[23] and is also concentrated in bile[24] which is excreted in feces.[25]
Ceruloplasmin is the majorcopper-carrying protein in the blood. Ceruloplasmin exhibits oxidase activity, which is associated with possible oxidation of Fe(II) into Fe(III), therefore assisting in its transport in theblood plasma in association with transferrin, which can carry iron only in the Fe(III) state.
Osteopontin is involved in mineralization in the extracellular matrices of bones and teeth.
Metalloenzymes all have one feature in common, namely that the metal ion is bound to the protein with onelabilecoordination site. As with allenzymes, the shape of theactive site is crucial. The metal ion is usually located in a pocket whose shape fits the substrate. The metal ioncatalyzes reactions that are difficult to achieve inorganic chemistry.
Inaqueous solution,carbon dioxide formscarbonic acid
This reaction is very slow in the absence of a catalyst, but quite fast in the presence of thehydroxide ion
A reaction similar to this is almost instantaneous withcarbonic anhydrase. The structure of the active site in carbonic anhydrases is well known from a number of crystal structures. It consists of azinc ion coordinated by threeimidazole nitrogen atoms from threehistidine units. The fourth coordination site is occupied by a water molecule. The coordination sphere of the zinc ion is approximatelytetrahedral. The positively-charged zinc ion polarizes the coordinated water molecule, andnucleophilic attack by the negatively-charged hydroxide portion on carbon dioxide proceeds rapidly. The catalytic cycle produces the bicarbonate ion and the hydrogen ion[2] as theequilibrium:
favouring dissociation ofcarbonic acid at biologicalpH values.[26]
Thecobalt-containingVitamin B12 (also known as cobalamin) catalyzes the transfer ofmethyl (−CH3) groups between two molecules, which involves the breaking ofC−C bonds, a process that is energetically expensive in organic reactions. The metal ion lowers theactivation energy for the process by forming a transient Co−CH3 bond.[27] The structure of thecoenzyme was famously determined byDorothy Hodgkin and co-workers, for which she received aNobel Prize in Chemistry.[28] It consists of a cobalt(II) ion coordinated to four nitrogen atoms of acorrin ring and a fifth nitrogen atom from animidazole group. In the resting state there is a Co−Csigma bond with the 5′ carbon atom ofadenosine.[29] This is a naturally occurringorganometallic compound, which explains its function intrans-methylation reactions, such as the reaction carried out bymethionine synthase.
Thefixation of atmospheric nitrogen is an energy-intensive process, as it involves breaking the very stabletriple bond between the nitrogen atoms. Thenitrogenases catalyze the process. One such enzyme occurs inRhizobiumbacteria. There are three components to its action: amolybdenum atom at the active site,iron–sulfur clusters that are involved in transporting the electrons needed to reduce the nitrogen, and an abundant energy source in the form ofmagnesiumATP. This last is provided by amutualistic symbiosis between the bacteria and a host plant, often alegume. The reaction may be written symbolically as
where Pi stands for inorganicphosphate. The precise structure of the active site has been difficult to determine. It appears to contain a MoFe7S8 cluster that is able to bind the dinitrogen molecule and, presumably, enable the reduction process to begin.[30] Some species of bacteria and archaea have also been shown to haveVanadium nitrogenases, which contain a VFe3S4 cluster and allows for an alternative pathway of nitrogen fixation in Molybdenum-deficient conditions.[31] The electrons are transported by the associated "P" cluster, which contains twocubical Fe4S4 clusters joined by sulfur bridges.[32]
Thesuperoxide ion,O−
2 is generated in biological systems by reduction of molecularoxygen. It has an unpairedelectron, so it behaves as afree radical. It is a powerfuloxidizing agent. These properties render the superoxide ion verytoxic and are deployed to advantage byphagocytes to kill invadingmicroorganisms. Otherwise, the superoxide ion must be destroyed before it does unwanted damage in a cell. Thesuperoxide dismutase enzymes perform this function very efficiently.[33]
The formaloxidation state of the oxygen atoms is −1⁄2. In solutions at neutralpH, the superoxide iondisproportionates to molecular oxygen andhydrogen peroxide.
In biology this type of reaction is called adismutation reaction. It involves both oxidation and reduction of superoxide ions. Thesuperoxide dismutase (SOD) group of enzymes increase therate of reaction to near the diffusion-limited rate.[34] The key to the action of these enzymes is a metal ion with variable oxidation state that can act either as an oxidizing agent or as a reducing agent.
In human SOD, the active metal iscopper, as Cu(II) or Cu(I), coordinatedtetrahedrally by fourhistidine residues. This enzyme also containszinc ions for stabilization and is activated by copper chaperone for superoxide dismutase (CCS). Otherisozymes may containiron, manganese ornickel. The activity of Ni-SOD involves nickel(III), an unusual oxidation state for this element. The active site nickel geometry cycles fromsquare planar Ni(II), with thiolate (Cys2 and Cys6) and backbone nitrogen (His1 and Cys2) ligands, tosquare pyramidal Ni(III) with an added axial His1 side chain ligand.[35]
Chlorophyll plays a crucial role inphotosynthesis. It contains amagnesium enclosed in achlorin ring. However, the magnesium ion is not directly involved in the photosynthetic function and can be replaced by other divalent ions with little loss of activity. Rather, thephoton is absorbed by the chlorin ring, whose electronic structure is well-adapted for this purpose.
Initially, the absorption of a photon causes anelectron to be excited into asinglet state of the Q band. Theexcited state undergoes anintersystem crossing from the singlet state to atriplet state in which there are two electrons with parallelspin. This species is, in effect, afree radical, and is very reactive and allows an electron to be transferred to acceptors that are adjacent to the chlorophyll in thechloroplast. In the process chlorophyll is oxidized. Later in the photosynthetic cycle, chlorophyll is reduced back again. This reduction ultimately draws electrons from water, yielding molecular oxygen as a final oxidation product.
Hydrogenases are subclassified into three different types based on the active site metal content: iron–iron hydrogenase, nickel–iron hydrogenase, and iron hydrogenase.[36]All hydrogenases catalyze reversibleH2 uptake, but while the [FeFe] and [NiFe] hydrogenases are trueredoxcatalysts, driving H2 oxidation and H+ reduction
the [Fe] hydrogenases catalyze the reversibleheterolytic cleavage of H2.
Since discovery ofribozymes byThomas Cech andSidney Altman in the early 1980s, ribozymes have been shown to be a distinct class of metalloenzymes.[37] Many ribozymes require metal ions in their active sites for chemical catalysis; hence they are called metalloenzymes. Additionally, metal ions are essential for structural stabilization of ribozymes.Group I intron is the most studied ribozyme which has three metals participating in catalysis.[38] Other known ribozymes includegroup II intron,RNase P, and several small viral ribozymes (such ashammerhead,hairpin,HDV, andVS) and the large subunit of ribosomes. Several classes of ribozymes have been described.[39]
Deoxyribozymes, also called DNAzymes or catalytic DNA, are artificial DNA-based catalysts that were first produced in 1994.[40] Almost all DNAzymes require metal ions. Although ribozymes mostly catalyze cleavage of RNA substrates, a variety of reactions can be catalyzed by DNAzymes including RNA/DNA cleavage, RNA/DNA ligation, amino acid phosphorylation and dephosphorylation, and carbon–carbon bond formation.[41] Yet, DNAzymes that catalyze RNA cleavage reaction are the most extensively explored ones. 10-23 DNAzyme, discovered in 1997, is one of the most studied catalytic DNAs with clinical applications as a therapeutic agent.[42] Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific),[43] the CA1-3 DNAzymes (copper-specific), the 39E DNAzyme (uranyl-specific)[44] and the NaA43 DNAzyme (sodium-specific).[45]
Calmodulin is an example of a signal-transduction protein. It is a small protein that contains fourEF-hand motifs, each of which is able to bind aCa2+ ion.
In anEF-hand loop protein domain, the calcium ion is coordinated in a pentagonal bipyramidal configuration. Sixglutamic acid andaspartic acid residues involved in the binding are in positions 1, 3, 5, 7 and 9 of the polypeptide chain. At position 12, there is a glutamate or aspartate ligand that behaves as abidentate ligand, providing two oxygen atoms. The ninth residue in the loop is necessarilyglycine due to the conformational requirements of the backbone. Thecoordination sphere of the calcium ion contains only carboxylate oxygen atoms and no nitrogen atoms. This is consistent with thehard nature of the calcium ion.
The protein has two approximately symmetrical domains, separated by a flexible "hinge" region. Binding of calcium causes a conformational change to occur in the protein. Calmodulin participates in anintracellular signaling system by acting as a diffusible second messenger to the initial stimuli.[46][47]
In bothcardiac andskeletal muscles, muscular force production is controlled primarily by changes in the intracellularcalciumconcentration. In general, when calcium rises, the muscles contract and, when calcium falls, the muscles relax.Troponin, along withactin andtropomyosin, is the protein complex to which calcium binds to trigger the production of muscular force.
Manytranscription factors contain a structure known as azinc finger, a structural module in which a region of protein folds around a zinc ion. The zinc does not directly contact theDNA that these proteins bind to. Instead, the cofactor is essential for the stability of the tightly folded protein chain.[48] In these proteins, the zinc ion is usually coordinated by pairs of cysteine and histidine side-chains.
There are two types ofcarbon monoxide dehydrogenase: one contains iron and molybdenum, the other contains iron and nickel. Parallels and differences in catalytic strategies have been reviewed.[49]
Pb2+ (lead) can replace Ca2+ (calcium) as, for example, withcalmodulin or Zn2+ (zinc) as withmetallocarboxypeptidases.[50]
Some other metalloenzymes are given in the following table, according to the metal involved.
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