Metalloenzymes
Metalloenzymes all have one feature in common, namely that the metal ion is bound to the protein with one labile coordination site. As with all enzymes, the shape of the active siteis crucial. The metal ion is usually located in a pocket whose shape fits the substrate. The metal ion catalyzes reactions that are difficult to achieve in organic chemistry.
Carbonic anhydrase
In aqueous solution, carbon dioxide forms carbonic acid
CO2 + H2O ⇌ H2CO3
This reaction is very slow in the absence of a catalyst, but quite fast in the presence of the hydroxide ion
- CO2 + OH− ⇌ HCO−
3
A reaction similar to this is almost instantaneous with carbonic anhydrase. The structure of the active site in carbonic anhydrases is well-known from a number of crystal structures. It consists of a zinc ion coordinated by three imidazole nitrogen atoms from three histidine units. The fourth coordination site is occupied by a water molecule. The coordination sphere of the zinc ion is approximately tetrahedral. The positively-charged zinc ion polarizes the coordinated water molecule, and nucleophilic attack by the negatively-charged hydroxide portion on carbon dioxide (carbonic anhydride) proceeds rapidly. The catalytic cycle produces the bicarbonate ion and the hydrogen ion[2] as the equilibrium
- H2CO3 ⇌ HCO−
3 + H+
favours dissociation of carbonic acid at biological pH values.
Vitamin B12-dependent enzymes
The cobalt-containing Vitamin B12 (also known as cobalamin) catalyzes the transfer of methyl (−CH3) groups between two molecules, which involves the breaking of C−C bonds, a process that is energetically expensive in organic reactions. The metal ion lowers the activation energy for the process by forming a transient Co−CH3 bond.[26] The structure of the coenzyme was famously determined by Dorothy Hodgkin and co-workers, for which she received a Nobel Prize in Chemistry.[27] It consists of a cobalt(II) ion coordinated to four nitrogen atoms of a corrin ring and a fifth nitrogen atom from an imidazole group. In the resting state there is a Co−C sigma bond with the 5′ carbon atom of adenosine.[28] This is a naturally occurring organometallic compound, which explains its function in trans-methylation reactions, such as the reaction carried out by methionine synthase.
Nitrogenase (nitrogen fixation)
The fixation of atmospheric nitrogen is a very energy-intensive process, as it involves breaking the very stable triple bond between the nitrogen atoms. The enzyme nitrogenase is one of the few enzymes that can catalyze the process. The enzyme occurs in Rhizobium bacteria. There are three components to its action: a molybdenum 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 of magnesium ATP. This last is provided by a symbiotic relationship between the bacteria and a host plant, often a legume. The relationship is symbiotic because the plant supplies the energy by photosynthesis and benefits by obtaining the fixed nitrogen. The reaction may be written symbolically as
where Pi stands for inorganic phosphate. 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.[29] The electrons are transported by the associated "P" cluster, which contains two cubical Fe4S4clusters joined by sulfur bridges.
Superoxide dismutase
The superoxide ion, O−
2 is generated in biological systems by reduction of molecular oxygen. It has an unpaired electron, so it behaves as a free radical. It is a powerful oxidizing agent. These properties render the superoxide ion very toxic and are deployed to advantage by phagocytes to kill invading microorganisms. Otherwise, the superoxide ion must be destroyed before it does unwanted damage in a cell. The superoxide dismutase enzymes perform this function very efficiently.[31]
2 is generated in biological systems by reduction of molecular oxygen. It has an unpaired electron, so it behaves as a free radical. It is a powerful oxidizing agent. These properties render the superoxide ion very toxic and are deployed to advantage by phagocytes to kill invading microorganisms. Otherwise, the superoxide ion must be destroyed before it does unwanted damage in a cell. The superoxide dismutase enzymes perform this function very efficiently.[31]
The formal oxidation state of the oxygen atoms is −1⁄2. In solutions at neutral pH, the superoxide ion disproportionates to molecular oxygen and hydrogen peroxide.
- 2 O−
2 + 2 H+ → O2 + H2O2
In biology this type of reaction is called a dismutation reaction. It involves both oxidation and reduction of superoxide ions. The superoxide dismutase (SOD) group of enzymes increase the rate of reaction to near the diffusion-limited rate. 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.
- Oxidation: M(n+1)+ + O−
2 → Mn+ + O2 - Reduction: Mn+ + O−
2 + 2 H+ → M(n+1)+ + H2O2.
In human SOD the active metal is copper, as Cu(II) or Cu(I), coordinated tetrahedrally by four histidine residues. This enzyme also contains zinc ions for stabilization and is activated by copper chaperone for superoxide dismutase (CCS). Other isozymes may contain iron, manganese or nickel. Ni-SOD is particularly interesting as it involves nickel(III), an unusual oxidation state for this element. The active site nickel geometry cycles from square planar Ni(II), with thiolate (Cys2 and Cys6) and backbone nitrogen (His1 and Cys2) ligands, to square pyramidal Ni(III) with an added axial His1 side chain ligand.
Chlorophyll-containing proteins
Chlorophyll plays a crucial role in photosynthesis. It contains a magnesium enclosed in a chlorin 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, the photon is absorbed by the chlorin ring, whose electronic structure is well-adapted for this purpose.
Initially, the absorption of a photon causes an electron to be excited into a singlet state of the Q band. The excited state undergoes an intersystem crossing from the singlet state to a triplet state in which there are two electrons with parallel spin. This species is, in effect, a free radical, and is very reactive and allows an electron to be transferred to acceptors that are adjacent to the chlorophyll in the chloroplast. 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.
Hydrogenase
Hydrogenases are subclassified into three different types based on the active site metal content: iron–iron hydrogenase, nickel–iron hydrogenase, and iron hydrogenase.[34] All hydrogenases catalyze reversible H2 uptake, but while the [FeFe] and [NiFe] hydrogenases are true redox catalysts, driving H2 oxidation and H+ reduction
- H2 ⇌ 2 H+ + 2 e−
the [Fe] hydrogenases catalyze the reversible heterolytic cleavage of H2.
- H2 ⇌ H+ + H−
Ribozyme and deoxyribozyme
Since discovery of ribozymes by Thomas Cech and Sidney Altman in the early 1980s, ribozymes has been shown to be a distinct class of metalloenzymes. Many ribozymes require metal ions in their active sites for chemical catalysis; hence they are called metalloenzymes. Additionally, metal ions are essential for the stabilization of ribozyme structure. Group I intron is the most studied ribozyme which has three metals participating in catalysis. Other known ribozymes include group II intron, RNase P, and several small viral ribozymes (such as hammerhead, hairpin, HDV, and VS). Recently, four new classes of ribozymes have been discovered (named twister, twister sister, pistol and hatchet) which are all self-cleaving ribozymes.
Deoxyribozymes, also called DNAzymes or catalytic DNA, are first discovered in 1994 and quickly emerged as a new class of metalloenzymes. Almost all DNAzymes require metal ions for their function; thus they are classified as metalloenzymes. Although ribozymes mostly catalyze cleavage of RNA substrates, 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. 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. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), the CA1-3 DNAzymes (copper-specific), the 39E DNAzyme (uranyl-specific) and the NaA43 DNAzyme (sodium-specific).
Signal-transduction metalloproteins
Calmodulin
In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. Six glutamic acid and aspartic acidresidues 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 a (bidentate ligand), providing two oxygen atoms. The ninth residue in the loop is necessarily glycine due to the conformational requirements of the backbone. The coordination sphere of the calcium ion contains only carboxylate oxygen atoms and no nitrogen atoms. This is consistent with the hard nature of the calcium ion.Calmodulin is an example of a signal-transduction protein. It is a small protein that contains four EF-hand motifs, each of which is able to bind a Ca2+ 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 an intracellular signaling system by acting as a diffusible second messenger to the initial stimuli.
Troponin
In both cardiac and skeletal muscles, muscular force production is controlled primarily by changes in the intracellular calcium concentration. In general, when calcium rises, the muscles contract and, when calcium falls, the muscles relax. Troponin, along with actin and tropomyosin, is the protein complex to which calcium binds to trigger the production of muscular force.
Transcription factors
Other metalloenzymes
Many transcription factors contain a structure known as a zinc finger, this is a structural module where a region of protein folds around a zinc ion. The zinc does not directly contact the DNA that these proteins bind to. Instead, the cofactor is essential for the stability of the tightly-folded protein chain.[46] In these proteins, the zinc ion is usually coordinated by pairs of cysteine and histidine side-chains.
There are two types of carbon monoxide dehydrogenase: one contains copper and molybdenum, the other contains nickel and iron. Parallels and differences in catalytic strategies have been reviewed.[47]
Pb2+ (lead) can replace Ca2+ (calcium) as, for example, with calmodulin or Zn2+ (zinc) as with metallocarboxypeptidases
Some other metalloenzymes are given in the following table, according to the metal involved.
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