Is O2 paramagnetic

First take a deep breath? - Our dangerous life as aerobes

• Hämerythrin - the oxygen transporter for marine invertebrates

Molecular structure of Hämerythrin (Hr)

Hr is in both the unloaded and the O2-charged state characterized crystallographically. Hr is a small protein with 113 amino acids that characteristically form a stack of four α-helices. This is shown using the example of Hämerythrin from Themiste dyscritum, a marine worm that was analyzed in a loaded and unloaded state. Two views of the column stack of the unloaded form are shown:

The active center has a free coordination point; the ligand equipment consists of five His side chains, a bridging hydroxo ligand, 1 Asp and 1 Glu:

The O binds to this position in the loaded state2-Molecule

Oxygen transport

Quantum chemical calculations show that the addition of oxygen is a redox process that is accompanied by a proton shift: the transfer of two electrons creates two iron (III) centers and one peroxide ion. The anion picks up the proton of the bridging hydroxo ligand and activates a hydrogen bond to the oxo ligand formed. When it is released, the reverse processes take place, iron (III) must be able to oxidize peroxide to oxygen. The standard potential in acidic solution for peroxide oxidation is 0.7 V, at pH 7 the result is 0.3 V. Without ligands, the iron (II, III) potential at pH 7 is approx. 0 V; oxygen release would be under these conditions not possible. Hr is another example of how a larger number of His ligands increases the electrochemical potential to such an extent that the required oxidizing power is available to the oxidized form.

• Myoglobin and Hemoglobin

Chemistry and Biochemistry of Hemoglobin (Hb) and Myoglobin (Mb)

The path of oxygen from the lungs to the mitochondria begins when it binds to hemoglobin. The transfer to takes place in the muscle cells Myoglobin, which has a larger binding constant for O2 as Hb has. From this the oxygen molecule is eventually attached Cytochrome c oxidase (CcO) transferred, at which the oxygen affinity reaches the greatest value. In the active center of CcO, the last enzyme in the respiratory chain, oxygen is reduced to water. The oxygen transport enzymes Hb and Mb are found in all vertebrates and many invertebrates. The ratio of Mb to Hb varies greatly between organisms. Particularly large amounts of Mb are used by lung-breathing animals living in the water in order to be able to store a lot of oxygen for long dives. Because of their high Mb level, whales and seals can stay under water for about half an hour. The ubiquitous sperm whale Mb in biochemistry. sperm whale myoglobin) is explained by the high availability of this protein.

Both transport enzymes are only able to bind oxygen if the iron center is in the + II oxidation state (deoxyMb and deoxyHb). Oxidation to the trivalent state with formation of metMb or metHb leads to deactivation.

A newly discovered function of the Mb and Hb family enzymes appears to be the elimination of NO. Mbs and Hbs can oxidize NO to nitrate. Starting from oxyMb or oxyHb, whose FeII-O2-Fragment itself here like a FeIII-O2•−Function behaves (see also below), the implementation can be according to

NO + O2•− → NO3

formulate, whereby metMb or metHb remains and then has to be converted back to the divalent initial state by a 1-e reduction.

Heme proteins appeared in evolution long before oxygen appeared in the atmosphere. The NO-binding properties have led to the hypothesis that the "primordial hemoglobin", whose formation is believed to have been around 3.5 billion years ago, served the NO metabolism and that the O2-Transport is a property that evolved later.


Mb is a 153 amino acid long protein that is dominated by eight α-helices. A hydrophobic pocket of the apoprotein contains an iron (II) protoporphyrin-IX complex (heme b) without covalent attachment of the porphyrin only via a His-Fe contact (see illustration at oxyMb). The His directly bound to iron is the “proximal” His (His93, often addressed as F8 [8th amino acid on α-helix F]). On the other side of the heme is the O2-Binding point. Another His is usually found here, the “distal” His (His64 or E7), which is located at a suitable distance from the heme in order to be able to build a hydrogen bond to H-bond acceptors as an N-H donor.

The picture shows deoxy-Mb at high resolution (1 Å). The distal His is found to be disordered, the water molecule drawn on the distal side is in an understaffed position. Due to the N ··· O distances between the water position and the distal His (2.76 Å), as well as the water position and one of the four porphyrin nitrogen atoms (2.74 Å), which are typical for hydrogen bonds, the disorder of the distal His can be understood: in part of the the crystal-forming Mb molecules lack the single water molecule on the distal side. This is understandable, since apart from the two N ··· O contacts there are no other binding possibilities for the water dipole - apart from His, the distal cavity is only lined with hydrophobic side chains (see below). The distal his only takes the left position in the presence of water. In the absence of the water molecule, the distal His moves to the right towards the protein edge, where it makes contact, not shown, with a water molecule outside the O2-Binding pocket can build up.

The iron (II) center in deoxyMb is in the S = 5/2 ground state, it is a high-spin center.


The structure of oxyMb is also available in high resolution. A look at the holoenzyme shows the position of the heme in a pocket formed by α-helices. The α-helices are labeled in the usual way:

An oxygen molecule is bound in the active center, the surrounding area of ​​which, with the exception of the distal His, consists only of hydrophobic side chains:

The interaction between the distal His and O2 becomes clearer when the observer looks into the active center from the left:

The distal His is the donor of a hydrogen bond to the terminal O atom of O.2Ligands. The N – H ··· O bond is not the cause of the angled Fe-O-O arrangement, but it uses this to establish the additional interaction and thus increases the binding constant for O2.

oxyMb is in the singlet ground state, which led Pauling to the conclusion that oxyMn is a low-spin iron (II) complex.


The stabilization of the O2Complex through the H-bond to the distal His seems to be the main reason for the relatively low CO affinity of Mb and Hb. While isolated heme CO approx. 105 binds times more effectively than O2, this ratio for Mb and Hb is lower by a factor of 1000. A 100-fold affinity for CO shows that carbon monoxide is still a poisonous gas if it is inhaled in unphysiologically high concentrations. However, the small amount of physiologically formed CO produced, for example, during the breakdown of heme loses the ability to block Mb or Hb at this ratio of binding constants.

Numerous structural analyzes of the binding of CO to Mb are available. The Leu29Trp mutant of Sperm Whale Mb, in which the hydrophobic space on the distal side is reduced by the mutation, was examined in detail. The CO adduct shows an only slightly bent Fe-C-O fragment:

In comparison with the oxyMb center, the distal His is now pushed to the right, the alignment of an N-H function to CO is not given. For a long time, the last two images were interpreted in such a way that CO pushes the distal His aside with considerable expenditure of energy, which would have resulted in a reduced binding constant. Current research has not confirmed this idea. The terminal O atom of O2 seems to be the much better H-bond acceptor compared to the O atom of carbon monoxide. The bond to CO is therefore not particularly destabilized by steric stress, the bond to O2 rather, it is particularly stable. This result suggests that nature has a higher negative charge on the terminal atom of O2-Ligands opposite the CO oxygen atom for a strong hydrogen bond. This conclusion leads to a comparison of the bonding in oxyMb and MbCO, which has one thing in common: both complexes are in the singlet ground state.

• hemocyanin (Hc)

Experiment 28-15: Oxidation of copper (I) by atmospheric oxygen.

Sub-steps like

2 CuII + O22− → 2 CuI. + O2


CuII + O2•− → CuI. + O2

In other words, the reduction of copper (II) to the usually inconsistent oxidation state + I by common oxidizing agents is inconceivable in "normal" aqueous chemistry. These reactions take place in the centers of Hc and CuZnSOD. The prerequisite is the coincidence of two enzyme characteristics: (1) the reactivity control introduced in the previous chapter through the stabilization of frontier orbitals and (2) the control of the electron balance.

Chemistry and Biochemistry of Hemocyanin

Hemocyanin is the oxygen transport molecule of snails and squids ("mollusks") as well as crabs, scorpions and spiders ("arthropods"). Hc single molecules form species-specific oligomers, the purpose of which is to build up cooperativity of the O2-Transport seems to lie (see Mb and Hb). In addition to myoglobin / hemoglobin and hemerythrin, hemocyanin is the third O2-Transporter found in organisms.

Type 3 copper proteins

Hemocyanin is a type 3 copper protein. This class of enzymes is characterized by dinuclear copper centers that activate O2 are able - in the sense of O2Transport, as an oxygenase or as an oxidase.

Molecular structure of hemocyanin

X-ray structure analyzes of the oxygenated and deoxygenated form of hemocyanin are available Limulus polyphemus, the horseshoe crab. The picture shows the structure of the protein, which is quite large with 628 amino acids, in a resolution of 2.4 Å):

In the active center loaded with oxygen, the distance between the copper atoms in the Cu bridged by a peroxo ligand isII2-Pair 3.6 Å:

In deoxygenated CuI.2At the center of horseshoe crab hemocyanin, the Cu-Cu distance is significantly larger at 4.6 Å:

2 CuII + O22− → 2 CuI. + O2 - Catalysis versus Thermodynamics?

The release of oxygen with the reduction of copper (II) to copper (I) is in stark contrast to laboratory experience and the position of the electrochemical potentials. Colorless ammine copper (I) solutions are rapidly and irreversibly oxidized to the familiar blue ammine copper (II) solutions by atmospheric oxygen. The fact that with Hc oxygen is released with formation of copper (I) is due to the combination of two enzyme-typical factors.

(1) In Hc, too, there is no regular square coordination of ligands, i.e. the arrangement that a strongly destabilized x2-Y2-Orbital created and as a result a low potential. In the case of the redox couple [CuI / II(NH3)4]+/2+ this is exactly the case. The potential of the ammine copper pair is therefore less than the Hc potential of approx. 0.3-0.4 V.

(2) An isolated binuclear center with the accessible oxidation states MI.2, MI.M.II and MII2 allows the conversion of a maximum of two electrons. The 4-electron reduction of O with a higher driving force2 (E.0'= 0.82 V; see Appendix II) cannot expire in such a center. At best, the 2-electron process oxygen / peroxide is possible - the potential of which coincides perfectly with the Hc potential (Appendix II).

The related and thermodynamically impossible reaction

CuII + O2•− → CuI. + O2

runs under analogous boundary conditions in copper-zinc-superoxide dismutase (CuZnSOD):

• Cytochrome c oxidase (CcO)

The chemistry of hemocyanin and CuZnSOD is determined by the definition of the electron balance based on the number of redox centers and their available oxidation states. The catalysis of a 2-electron oxidation by a mononuclear copper protein must therefore be irritating. Is copper (III) to be considered in galactose oxidase?

Chemistry and biochemistry of cytochrome c oxidase (CcO)

CcO is the last enzyme in the respiratory chain. CcO is reduced by four equivalents of cytochrome c in order to subsequently reduce an oxygen molecule to water in one step. The energy gained is stored as a proton gradient. The large number of four electrons for the reaction

O2 + 4 e + 4 H.+ → 2 H2O

places special demands on the structure of the active center.

Structure of the heme a3-CuB.Center in cytochrome c oxidase (CcO)

CcO consists of 26 protein chains with a total of 3614 amino acids. A structural analysis of bovine heart CcO in the oxidized state with 1.8 Å resolution is available. The O2-reducing heme-a3-CuB.Center occurs together with another heme-a in two protein chains. One of these two chains (A), consisting almost exclusively of α-helices, is shown, in addition, chain B, which is rich in leaflet sections, is in the upper part of the illustration. Between A and B there is an electron-conducting CuA.-Center:

The protein shown extends through the inner mitochondrial membrane. The lower part protrudes into the interior of the organelle, the upper part protrudes into the transmembrane space between the inner and outer mitochondrial membrane. The electrons transported by Cyt c are taken from the CuA.Center accepted and passed on to the heme a center. The flow of electrons ends at the oxygen-reducing heme-a3-CuB.-Center. The four protons of the gross equation come from inside the mitochondrion (in the picture below).

When heme a3-CuB.Center, the area around the copper atom is particularly noticeable. This consists of three His ligands, one of which has a direct bond to a Tyr residue:

To reduce an O2Molecule, it is bound between the two metal centers and charged with 4 electrons in one step.

• hydrogen peroxide, H2O2

Experiment 18-1: H.2O2 arises from the combustion of hydrogen when the flame is quenched

Experiment 3-16: Catalytic H.2O2-Decomposition by platinum

Experiment 3-15: Catalytic H.2O2-Decomposition by potato catalase

New experiment: catalytic H2O2-Decomposition by bovine serum catalase

• heme catalase

The reactivity of a heme center can be controlled by the choice of additional ligands. Block in cytochrome c two further ligands the iron atom for a substrate bond, so that only the exchange of electrons is possible. In the case of heme catalase, an anionic ligand leads to the stabilization of higher oxidation states than is possible with Mb and Hb.

Heme catalase is used to disproportionate H.2O2that is produced by SODs, for example:

H2O2 → H2O + ½ O2

In the case of heme catalase, the biological functional unit is probably the tetramer of the asymmetrical unit shown:

The protein structure bears little resemblance to myoglobin, yet the active site is a startlingly conservative variation on the Mb center. The cofactor of the apoprotein is a heme b. As in Mb and Hb, above the heme there is a space that is hydrophobically lined with phenylalanine side chains and also contains a “distal” His. In its vicinity there is a water molecule at an N ··· O distance of 2.65 Å. 2.42 Å away from the water molecule is an O atom of an HO bound to iron (III) (?)2Ligands. The most striking difference to metMb is the replacement of the proximal His by a tyrosinato ligand, which represents the trivalent level of iron as a reduced form of the redox couple FeIII/ FeIVPor•+ stabilized.

The H2O2-Disproportionation requires a 2-electron redox process. In the first step the FeIII-The rest form of the enzyme is oxidized by two electrons, an Oxoferryl species is created, which then returns to the rest form:

Por-FeIII + H2O2 → Por•+-FeIV= O + H2O

Por•+-FeIV= O + H2O2 → Por-FeIII + H2O + O2

• Copper-zinc superoxide dismutase (CuZnSOD)

Chemistry and biochemistry of CuZnSOD

The importance of superoxide removal has already been discussed in connection with MnSOD. The phylogenetic relationships are reflected in the occurrence of the individual SODs. FeSOD occurs in prokaryotes. MnSOD, which probably developed from FeSOD, also occurs in prokaryotes, but also in the mitochondria of higher living beings. Finally, CuZnSOD - as the latest enzyme in which two metals are combined that only became available in a sulfur-free oxygen world - is found in the cytosol of all eukaryotes. The concentration here is approx. 10−5 mol L−1. In it is calculated that due to the concentration and the activity of the enzyme, the lifetime of superoxide radicals by a factor of 1010 is lowered.

Non-blue copper proteins

CuZnSOD belongs to the type 2 copper proteins, also called "non-blue" copper proteins. The UV / Vis spectra essentially correspond to those of “normally” coordinated copper (II) complexes; the strong charge-transfer transition of the blue copper proteins is missing. The galactose oxidase (GO) treated below also belongs to the type 2 centers.

The molecular structure of CuZnSOD

The structure of human CuZnSOD was determined with a resolution of 1.8 Å:

The specialty of the CuZnSOD center is a histidinato ligand bridging between zinc (left) and copper (right). The distance between the copper (II) atom and the bridging N.His (dashed line) is approximately 2 Å in the oxidized form and more than 3 Å in the reduced form. The structural analysis shows a mean value of approx. 2.6 Å, the cause of which the authors state in part to be photoreduction of the oxidized resting form during X-ray exposure:

Catalysis cycle

According to a current proposal in, superoxide is protonated on the way to the active site and reaches the central copper atom as HO2 (pKA.(HO2) = 4.8), where it takes the place of the aqua ligand (other authors usually formulate it with the superoxide anion and the two required protons are added separately). The water molecule found on copper (II) in the structural analysis is not taken into account in the following scheme:

The numbering applies to the human enzyme described in. Bovine erythrocyte CuZnSOD, in which copper is coordinated by His residues 44, 46, 61, and 118, is widely studied.

The catalytic cycle emphasizes the statement that the electron difference in the available oxidation states determines the reaction that takes place. Since zinc is redox-inactive - its importance is seen in the increase in the acidity of the bridging His - only 1-electron steps are possible. This is exactly what superoxide disproportionation demands.

The electrochemical potential is also in the necessary range. As a reminder: while the potential of an oxygen transporter must correspond to the potential of the redox process used, the potential of a disproportionation catalyst must lie between the potentials of the two catalyzed individual steps.

• Appendix: Electrochemical potentials of oxygen species at pH 7

The following graphic shows electrochemical potentials at pH 7 and activity 1. The numerical values ​​are taken from: D. M. Kurtz jr .: Dioxygen-binding proteins. CCC 8, 229-260 (230).

The number of electrons transferred is color-coded:
black: 1 electron,
blue: 2 electrons,
green: 3 electrons,
red: 4 electrons.