Biocomplexes

Biocomplexes

Metal binding ability of biomolecules

Donor atoms in biological systems hetero atoms with electron pairs: O, N, S, (Se)

- oxygen donor ligands:alcohols: R-OH, (e.g. carbohydrates)ethers: R-O-R (e.g. carbohydrates)carbonyl compounds: -CO- (e.g. proteins) phenols: Ar-OH (e.g. polyphenols)carboxyilic comounds: -COOH (e.g. carboxylic acids, amino acids)O-heterocycles: (furane, ..)

- nitrogen donor ligands:amines: R-NH2 (e.g. amino acids)amides: -CONH- (e.g. peptide bond)N-heterocycles: (pyrrole, pyridine, imidazole,...)

- sulphur donor ligands:thiol compounds/disulphides: RSH/R-S-S-R (e.g. cysteine-cystine)thioethers: R-S-R (pl. methionine)sulphur containing heterocycles: (pl. tiophene,..)

Main groups of bioligands and their complex forming properties

- Coordination chemistry of amino acids:

H2N CH C

CH3

OH

O a/ amino acids without other functional group:Gly, Ala, Phe,....5-membered chelate ring (NH2,COO-)-coordination

H2N CH C

CH2

OH

O

N

NH

H2N CH C

CH2

OH

O

SH

b/ amino acids with 3 functional groups:

formation of 2 chelate rings

alcoholic-OH: Ser, Thr carboxylate: Asp, Glu,amide: Asn, Gln,amine: Lys, Orn,

thio: Cys, Met,

imidazole-N: HisHis

Cys

1. ML, ML2,..................MLn

(NH2, CO)- or COO— coordination, the latter is less favoured

R1 CH C NH CH C NH CH C NH CH COOH

H2N O R2 O R3 O R4

2. ML MLH-1 MLH-2 MLH-3

(NH2,CO) (NH2,N-,CO) (NH2,N-,N-,CO) (NH2,N-,N-,N-)

Metal ion promoted amide deprotonation and coordinationCu(II), Ni(II), Pd(II), ……...Formation of joint chelate systems is preferred

- Coordination chemistry of peptides

M2+

NH2 N-

N- N-

CH

C

CH C

R1

O

R2 O

CH

C

R3

O

CHCOO-

R4MH-3L

1. Coordination of the Peptide skeletone in 4N complexes(joint, five membered chelates)

2. Site specific role of R1, R2,.. Side chains (His, Cys)

- coordination chemistry of proteins: (the ligands have predetermined structure)

Chelate formation has only secondary importanceIndependent coordination of the side-chain donor atoms e.g. structure of carboanhydrase:

His 119

His 96

His 94

H2O

Zn2+

Glu 117

His 119

Thr 199

His 96

His 94

His 64

Glu 106

H2O

Gln 92

Zn2+

CarboanhydraseCarboanhydrase

CarboanhydraseCarboanhydrase

- coordination chemistry of proteins: the proteins significantly change their structure due to

metal ion coordination e.g. development of the zinc-finger structure:

N

NN

N

NH2

O

HOH

HH

HH

OP-O

O-

O

- Coordination chemistry of nucleic acids and their building blocks

AMP Potential binding sites:- nucleic bases: purine (adenine/guanine) pirimidine (cytosine/uracil)

- sugar: ribose or deoxy-ribose

- phosphate

Characteristics: no chelate formation → macrochelate or loop formation is possible

Metal ion selectivity:Nucleo bases: „soft” metal ions: e.g. Pt biological rolePhosphate: „hard” metal ions: e.g. Ca, Mg, Al,...

Difference in DNA/: cis-OH groups → (chelate formation)

- Metalloporphyrins and related compounds

Composition/Structure:N-donor containing, natural macrocyclescomplex forming:

porphine, chlorin-, corrin-, corphin-ring containing compounds

4 pyrrole (or indole) rings in joint conjugated or partly conjugated system (most important: tetrapyrrole compounds)

Stability of their metal complexes:Differs from the usual stabilty sequencesRatio of the inner hole and the size of the metal ion has

primary importance.Metallation is a catalytic process.

Metalloporfirinek

N

N

N

N

M2+ + H2P MP + 2H+

Stabilitási sor: Mg(II) < Zn(II) < Cu(II) < Fe(II) < Ni(II) < Pd(II) < Pt(II)

N = 4 (Ni(II), Pt(II), Pd(II)) - minden koordinációs hely foglaltN= 6 (Fe(II), Co(II), Mg(II), Zn(II) - axiális kötõhely

M

Stability trend

Axial binding sitesAll coordination sites are saturated

- Metalloporphyrins

Complexation with other bioligands

- carbohydrates: numerous –OH groupsin acidic/neutral milieau weak metal ion bindersmight be suitable for administration of metal ions

(medicinal application)-derivatives: aldolic acids amino sugars,- lipids, oils: contain few functional groups

very weak complexation

Summary:Strongest metal ion binders: proteins and macrocyclic ligands

→ enzymes, coenzymes, prosthetic groups

Further interactions:redox and acid-base interactions with vitamines,

hormones and many other biomolecules

Ellenőrző kérdések

1. Mi az alapvető különbség az aminosavak és az oligopeptidek fémionkötő képessége között?

2. Mi a jellemző a fehérjék fémion koordinációjára?3. Mely oldallánc donorcsoportok játszanak meghatározó

szerepet a fémionok megkötésében a fémtartalmú fehérjékben? Adjon példákat!

4. Jellemezze a nukleinsavak és nukleotidok fémion koordinációját!

5. Miért gyenge a szénhidrátok fémion megkötő képessége? Hogyan fokozható ez?

6. Milyen biomolekulákban fordul elő a tetrapirrol váz? Röviden jellemezze ezeket!

Basic coordination chemistry

1. Basic terms: (see advanced inorganic chemistry)formation: Lewis acid-base reaction

ligand: base metal ion: acidequilibrium:stepwise (successive)kinetics: labile and inert complexescoordination number: 2 – (10)

with essential elements: (2), 4, 6coordination geometry: 4 – tetrahedral/square planar,

6 – octahedral effects of complex formation:

colour and magnetic feature(crystal field theory)changes in the redox potential:(Fe3+/Fe2+ and Cu2+/Cu+ systems)

2. Complex formation in biological systems(multi)component systems

pM + sH + qA + rB MpAqBrHs

Types of complexes:biner (parent): MA, ........... MAn

polynuclear: MnA (A – bridging ligand)protonated: MHA (multifunctional ligand)

MH-1A (coordinated H2O – hydroxocomplex or„A”-ligand metal ion induced deprotonation

terner (mixed ligand/metal complexes): MAB

Reactions of complexes:acid-base: liberation/uptake of protonsredox: redox reactions of the metal ion or coordinated ligandsother reactions of coordinated ligands: templates,

changes in conformation, etc.

The preferential coordination of metal ions to bioligands/biodonors generally

can be well explained by the hard-soft acid-base theory.

Hard-soft classification of the biologically important metal ions and ligands

HardAcids

Soft

LowLowHigherSmallIonic bond

PolarizabilityElectronegativetyPositive chargeSizeChemical interactions

HighHighLowerLargeCovalent or -bound

HardBases

Soft

LowHighHigherSmallIonic bond

PolarizabilityElectronegativetyNegative chargeSizeChemical interactions

HighLowLowerLargeCovalent or -bound

The basic features of hard and soft acids and bases

The general rule is that hard acids makes strong interactions with hard bases and soft acids with soft bases. Accordingly, hard acids, like Ca2+, Fe3+, Al3+ prefer the oxygen-, fluorine- and partly N donor atoms, while the soft acids, like Cu+, Pt2+, Hg2+ and Cd2+ prefer the sulphur, phosphorous, and iodine donors in forming coordination compounds.

Because of the different „shape” of the d orbitals the energy degeneracy of the orbitals is lifted. Orbitals in a given geometry directed towards the ligands (electron pairs) will have higher energy and will be able to form bonds, while those not directed to ligand lone electron pairs will occupy lower energy and will be able to form bonds.

Splitting of d-orbitals in different fields

When the electrons will be redistributed among the d orbitals the sequence will be determined by the relation between the crystal field splitting energy () and the spin pairing energy (P). The figure shows the possible electron configurations in the case of octahedral geometry.

Distribution of electrons on the d-orbitals

Common geometries for 2-6 coordinate metal ions

CationNa+

K+

Mg2+

Ca2+

Mn2+ (d5)

Mn3+ (d4)Fe2+ (d6)

Fe3+ (d5)

Ca2+ (d7)

Ni2+ (d8)

Cu1+

(d10)

Cu2+ (d9)

Zn2+

(d10)

Coord number66-866-86

646

46

4

6

4

64

4

6

4

5

GeometryOctahedralFlexibleOctahedralFlexibleOctahedral

TetrahedralTetrahedralOctahedral

TetrahedralOctahedral

Square planar

Octahedral

Square planar

OctahedralTetrahedral

Tetrahedral

Suqare planar

Octahedral

Tetrahedral

Square pyramidal

Biologic ligandsO, ether, hydroxyl, carboxylateO, ether, hydroxyl, carboxylateO, carboxylate, phosphateO, carboxylate, carbonyl (phosphate)O, carboxylate, phosphateN, imidazole NO, carboxylate, phosphate, hydroxydeS, thiolateO, carboxylate, alkoxide, oxide, phenolate, N, imidazole N, porphyrinS, thiolateO, carboxylate, alkoxide, oxide, phenolate, N, imidazole N, porphyrinS, thiolateN, imidazole NO, carboxylateN, imidazole NS, thiolateN, imidazole N, polypirrol (F-430)RareS, thiolate, thioetherN, imidazole NS, thiolate, thioetherN, imidazole NO, carboxylateN, imidazole NO, carboxylateN, imidazole NO, carboxylate, carbonylS, thiolateN, imidazole NO, carboxylate, carbonylN, imidazole N

Standard redox potential of several iron complexes

The following basic conclusions can be drawn from the data in the above Table: (i) a decrease in the redox potential means stabilisation of the FeIII state as compared to FeII. That is, in the presence of hydroxide, cyanide, or oxalate ions FeII can be oxidised to FeIII or FeIII can hardly be reduced to FeII. In basic solution weak oxidising agents, like molecular oxygen, can oxidise FeII to FeIII. (ii) an increase in the redox potential means that the FeII state is stabilised as compared to the FeIII. In the presence of 2,2’-dipyridyl or 1,10-phenantrolin FeII can be oxidised to FeIIIonly by very strong oxidising agents.

Redox potential of several copper complex

In aqueous solution standard redox potential of the Cu2+ + e- Cu+ system is +,167 V. This suggests that in the absence of any complexing agents the Cu2+ ions are more stable. Depending on the type of the ligands either the CuII, or the CuI state can stabilised. From the data in the above Table it can be concluded that the lower oxidation state of copper the CuI can be stabilised in the presence of aromatic N-compounds (pyridin, imidazol), furthermore sulphur containing compounds, while the CuII state can be stabilised by N or O containing ligands (e.g. amino acids, such as alanin).

Redox systemCu(alanine)2

2+ + e- → Cu(alanine)2+

Cu(glycine)22+ + e- → Cu(glycine)2

+

Cu2+ + e- → Cu+

CuL22+ + e- → CuL2

+

Cu(pyridine)22+ + e- → Cu(pyridine)2

+

Cu(imidazole)22+ + e- → Cu(imidazole)2

+

Cu(CN)2 + e- → Cu(CN)-

E0(V)-0.130-0.160+0.167+0.243+0.270+0.345+1.103

(L = 2-methyl-thioethyl-amine)

Exchange rates for inner sphere water molecules

In general, it can be said that the exchange rate is higher for the less highly charged less strongly bound metal ions, than the more highly charged metal ions. The highly inert first row Cr3+ és Co3+ metal ions practically have no biological importance. Similarly, the second and third row transition metal ions have less biological importance too.

pK of various ligands in the absence and presence of biologically relevant metal ions

Ligand and reaction Metalion

lg K

Methods in bioinorganic chemistry I.

1. Determination of the composition:- solid phases: elemental analysis- solution equilibrium studies (determination of the stability constants)

(potentiometry, other techniques → measurements of some of the free components)

- mass spectrometry (ESI-MS)

2. Kinetics:- slow substitution reactions (inert complexes):

classical analytical methods → measurements of the concentration of some of the components)

- fast substitution reactions (labile complexes):stopped flow, T- jump and realxation methods (e.g. NMR).

Physical methods in bioinorganic chemistry II.

3. Structural investigating methods:a/ optical spectroscopic methods:

- UV-Vis spectrophotometry(d-d transitions, charge transfer (CT) and ligand transitions)- circular dicroism (CD, optical active compounds)

b/ magnetic methods:- magnetic momentum (low and high spin complexes)- ESR (EPR) spestroscopy (e.g. Cu2+, Mn2+, VO2+,...)- NMR spectroscopy (1H, 13C, ligand peaks)

multinuclear: 15N, 17O, 19F, 27Al, 51V, 113Cd, 195Pt,..

c/ other methods:- Mössbauer spectroscopy: (pl. Sn, Fe...)- X-ray diffraction- mass spectrometry

Enzymes I.

1. Definition: Catalysts of biological systems

2. Importance: chemical process: A Bthe catalyst accelerates the time reaching the equilibrium state,but equilibrium concentrations [A], [B] do not change.

Biological system: stationer equilibrium (steady state) → A B C D ............... →

E1 E2 E3

[A]stat, [B]stat, [C]stat = f(E1,E2,…En)The concentrations measured at the stationer equilibrium stateare not the same as those in the thermodynamic equilibrium, butdepend upon the reaction rates (enzymes). → The system is in continous change, it „goes” to the equilibrium state.

Enzymes II.

3. Naming enzymes:process + asee.g. peptidase (hydrolysis of peptides)

carboxypeptidase (from the direction of the C-terminus)( enzyme catalogue: EC x.y.z.w.e.g. EC 6.3.1.2. glutaminsynthetase)

4. Classification of Enzymes:Class type pf reactionoxidoreductases redox reactionstransferases transfer of atom or group of atomshydrolases hydrolysisliasese non hydrolytic cleavegeligases linking groups togetherisomerases isomeric trasformation

5. Selectivity: usually highfunction specificity: catalysis of a given reaction type (e.g. carboxy-

peptidase)substrate specificity: catalysis of a given range of substrates within

the function specificity (e.g. carboxypeptidase A: only with substrates with hydrophobic side chain)

6. Composition: simple or complex proteinssimple protein: M ≥ 10.000 ( ≥ 100 amino acid)complex protein: protein + prosthetic group or coenzymeprosthetic group: reversible non separable

(e.g. hem, biotin, metal ions, e.g. Cu, Fe,..)coenzyme: existing biomolecule

(e.g. NAD, ATP, Mo-co, metal ions, e.g. Ca, Mg, Zn,...)[ribozymes: RNA based enzymes]

Enzymes III.

Enzymes IV.

7. Metalloenzymes: ~ 30 % of the enzymes

- Binding of the metal ions:prosthetic group (e.g. hem, Fe, Cu,...)coenzyme: (e.g. B12, Mo-co, Ca, Mg, Zn, .....)

- Role of the metal ions:active centre: direct interactions with the substrate.

characteristics: distorted and unsaturated coordination geometry (high energy state)(e.g. carbonanhydrase (Zn), carboxypeptidase(Zn),superoxide dismutase(Cu))

structure maker (stabilizer): the metal ion fixes the conformationof the protein, usually saturated coordination geometry.(e.g. alcohol dehydrogenase(Zn), superoxide dismutase(Zn)

Kinetics of enzyme reactions I.

1. Kinetic model: Michaelis-MentenThesis: The initial rate (vo) of the enzyme catalysed reactions show saturation curve in the function of the concentration of the substrate ([S]).

vo

Vmax

Vmax/2

KM [S]

Kinetics of enzyme reactions II.

The general equation describing the previous function:

SbSa

vo

a = Vmax b = KM

To explain this the stationary equilibrium(steady state) has to be applied:

E + S ES → E + P k1

k-1

k2

i.e. the product (P) is formed through an enzyme-substrate complex (ES).If the concentration of the substrate is sufficiently high ([S] >> [Eo])Concentration of ES is constant in time, i.e. the rate of its formation and decomposition equals:

SEkdtESd

1 ES)kk(dtESd

21

Kinetics of enzyme reactions III.

Assuming the stationary equilibrium:

dtESd

dtESd

SEk1 ES)kk( 21

Taking into account the equations expressing of total enzyme concentrations and the rate equation of product formation:

ESEE o ESkv 2o

By substituting and rearranging the above equations:

1

21

o2o

kkk

S

SEkv

The initial rate is at maximum, when [ES] = [Eo]Vmax = k2·[Eo], i.e. in the rate equation:

maxo2 VEka and b = 1

21M k

kkK

Inhibition of enzyme reactions I.

1. Reversible inhibition: (I: inhibitor, E: enzyme, S: substrate)

a/ competitiv inhibition: the inhibitor and the substrate compete for the active

centres of the enzymeI → E but I → ES KM increases, but Vmax ≠ f(I)

b/ non-competitiv inhibition:the inhibitor interacts also with the enzyme-substrate complex (a Vmax also decreases)

Inhibition of enzyme reactions II.

2. Irreversible inhibition:

Strong (covalent) interactions between the inhibitor and theEnzyme, which can not be cleaved by the substrate.

If [I] > [E] activity of the enzyme can be completely stopped.

Heavy metal ions and chelators can be frequent inhibitors.

Mechanism of enzyme reactions (metalloenzymes)

Mechanism of the metalloenzyme catalysed reactions can be grouped in three main types:(L: substrate, activator or inhibitor)

1. Ligand bridged (or substrat bridged)

There is no direct interaction between the metal ion and the enzyme,

But it is necessary for the activation of the enzyme-substrate complex.

2. Metal bridgeda/ The substrate is in direct interaction only with the metal ion(occurs very rarely)

b/ The substrate is in interactions with the enzyme and the metal ion too (mpost frequent case)

3. Enzyme bridgedThere is no direct interaction between the metal ion and the substrate,But binding of the metal ion to the protein is necessary to the catalytic activity (structure maker)

(e.g. alcohol dehydrogenase)