Metalloprotein

Metalloprotein is a generic term for a protein that contains a metal ion cofactor. A large proportion of all proteins are part of this category. For instance, at least 1000 human proteins contain zinc-binding protein domains although there may be up to 3000 human zinc metalloproteins.

Abundance

It is estimated that approximately half of all proteins contain a metal. In another estimate, about one quarter to one third of all proteins are proposed to require metals to carry out their functions. Thus, metalloproteins have many different functions in cells, such as storage and transport of proteins, enzymes and signal transduction proteins, or infectious diseases.
Most metals in the human body are bound to proteins. For instance, the relatively high concentration of iron in the human body is mostly due to the iron in hemoglobin.

	Liver	Kidney	Lung	Heart	Brain	Muscle
Mn	138	79	29	27	22	<4-40
Fe	16,769	7,168	24,967	5,530	4,100	3,500
Co	<2-13	<2	<2-8	---	<2	150
Ni	<5	<5-12	<5	<5	<5	<15
Cu	882	379	220	350	401	85-305
Zn	5,543	5,018	1,470	2,772	915	4,688

Coordination chemistry principles

In metalloproteins, metal ions are usually coordinated by nitrogen, oxygen or sulfur centers belonging to amino acid residues of the protein. These donor groups are often provided by side-chains on the amino acid residues. Especially important are the imidazole substituent in histidine residues, thiolate substituents in cysteine residues, and carboxylate groups provided by aspartate. 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 deprotonated amides and the amide carbonyl oxygen centers. Lead binding in natural and artificial proteins has been reviewed.
In addition to donor groups that are provided by amino acid residues, many organic cofactors function as ligands. Perhaps most famous are the tetradentate N₄ macrocyclic ligands incorporated into the heme protein. Inorganic ligands such as sulfide and oxide are also common.

Storage and transport metalloproteins

These are the second stage product of protein hydrolysis obtained by treatment with slightly stronger acids and alkalies.

Oxygen carriers

, which is the principal oxygen-carrier in humans, has four subunits in which the iron ion is coordinated by the planar macrocyclic ligand protoporphyrin IX and the imidazole nitrogen atom of a histidine residue. The sixth coordination site contains a water molecule or a dioxygen molecule. By contrast the protein myoglobin, found in muscle cells, has only one such unit. The active site is located in a hydrophobic pocket. This is important as without it the iron would be irreversibly oxidized to iron. The equilibrium constant for the formation of HbO₂ is such that oxygen is taken up or released depending on the partial pressure of oxygen in the lungs or in muscle. In hemoglobin the four subunits show a cooperativity effect that allows for easy oxygen transfer from hemoglobin to myoglobin.
In both hemoglobin and myoglobin it is sometimes incorrectly stated that the oxygenated species contains iron. It is now known that the diamagnetic nature of these species is because the iron atom is in the low-spin state. In oxyhemoglobin the iron atom is located in the plane of the porphyrin ring, but in the paramagnetic deoxyhemoglobin the iron atom lies above the plane of the ring. This change in spin state is a cooperative effect due to the higher crystal field splitting and smaller ionic 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 the carboxylate side chains of a glutamate and aspartate and five histidine residues. The uptake of O₂ by hemerythrin is accompanied by two-electron oxidation of the reduced binuclear center to produce bound peroxide. The mechanism of oxygen uptake and release have been worked out in detail.
Hemocyanins carry oxygen in the blood of most mollusks, and some arthropods such as the horseshoe crab. They are second only to hemoglobin in biological popularity of use in oxygen transport. On oxygenation the two copper atoms at the active site are oxidized to copper and the dioxygen molecules are reduced to peroxide,.
Chlorocruorin is an oxygen-binding hemeprotein present in the blood plasma of many annelids, particularly certain marine polychaetes.

Cytochromes

and reduction reactions are not common in organic chemistry as few organic molecules can act as oxidizing or reducing agents. Iron, on the other hand, can easily be oxidized to iron. This functionality is used in cytochromes, which function as electron-transfer vectors. The presence of the metal ion allows metalloenzymes to perform functions such as redox reactions that cannot easily be performed by the limited set of functional groups found in amino acids. The iron atom in most cytochromes is contained in a heme group. The differences between those cytochromes lies in the different side-chains. For instance cytochrome a has a heme a prosthetic group and cytochrome b has a heme b prosthetic group. These differences result in different Fe²⁺/Fe³⁺ redox potentials such that various cytochromes are involved in the mitochondrial electron transport chain.
Cytochrome P450 enzymes perform the function of inserting an oxygen atom into a C−H bond, an oxidation reaction.

Rubredoxin

is an electron-carrier found in sulfur-metabolizing bacteria and archaea. The active site contains an iron ion coordinated by the sulfur atoms of four cysteine residues forming an almost regular tetrahedron. Rubredoxins perform one-electron transfer processes. The oxidation state of the iron atom changes between the +2 and +3 states. In both oxidation states the metal is high spin, which helps to minimize structural changes.

Plastocyanin

Plastocyanin is one of the family of blue copper proteins that are involved in electron transfer reactions. The copper-binding site is described as distorted trigonal pyramidal. The trigonal plane of the pyramidal base is composed of two nitrogen atoms from separate histidines and a sulfur from a cysteine. Sulfur 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 than Cu−S₂.
The elongated Cu−S₂ bonding destabilizes the Cu form and increases the redox potential of the protein. The blue color is due to the Cu−S₁ bond where S to Cu charge transfer occurs.
In the reduced form of plastocyanin, His-87 will become protonated with a pK_a of 4.4. Protonation prevents it acting as a ligand and the copper site geometry becomes trigonal planar.

Metal-ion storage and transfer

Iron

is stored as iron in ferritin. The exact nature of the binding site has not yet been determined. The iron appears to be present as a hydrolysis product such as FeO. Iron is transported by transferrin whose binding site consists of two tyrosines, one aspartic acid and one histidine. The human body has no controlled mechanism for excretion of iron. This can lead to iron overload problems in patients treated with blood transfusions, as, for instance, with β-thalassemia. Iron is actually excreted in urine and is also concentrated in bile which is excreted in feces.

Copper

Ceruloplasmin is the major copper-carrying protein in the blood. Ceruloplasmin exhibits oxidase activity, which is associated with possible oxidation of Fe into Fe, therefore assisting in its transport in the blood plasma in association with transferrin, which can carry iron only in the Fe state.

Calcium

Osteopontin is involved in mineralization in the extracellular matrices of bones and teeth.

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 site is 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
This reaction is very slow in the absence of a catalyst, but quite fast in the presence of the hydroxide ion
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 proceeds rapidly. The catalytic cycle produces the bicarbonate ion and the hydrogen ion as the equilibrium:
favouring dissociation of carbonic acid at biological pH values.

Vitamin B₁₂-dependent enzymes

The cobalt-containing Vitamin B₁₂ catalyzes the transfer of methyl 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. The structure of the coenzyme was famously determined by Dorothy Hodgkin and co-workers, for which she received a Nobel Prize in Chemistry. It consists of a cobalt 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. This is a naturally occurring organometallic compound, which explains its function in trans-methylation reactions, such as the reaction carried out by methionine synthase.