Cytochrome P450

Figure 1 The fold of cytochrome P450s is highly conserved and shown in a ribbon representation (distal face). Substrate recognition sequence (SRS) regions are shown in black and labeled. α-Helixes mentioned in the text are labeled with capital letters.

P450 catalyti cycle

Figure 3 Crystal structure of the oxy complex of wild-type CYP101 (pdb code 1dz8) that catalyzes the 5-exo hydroxylation of camphor. Oxygen binding induces a rotation of the amide of Asp251, which ensues binding of two new water molecules, Wat901 and Wat902.

• P450 catalytic mech focuses on state of heme iron and O
• Steps:

1. Oxygen binds reduced heme iron. Formation of oxygenated heme Fe2+-OO or Fe3+-OO-
2. One-electron reduction of complex to a ferric peroxo state Fe3+-OO2-, which is earily protonated to form hydroperoxo Fe3+-OOH- 
3. 2nd protonation of latter Fe3+-OOH- complex at distal oxygen atom to form unstable transient Fe-OOH2. Followed by heterolytic scission of O-O bond. Water mol released
4. Reactions of remaining higher valent pophyrin metal-oxo complex, a ferryl-oxo ð-cation porphyrin radical and referred to as “Compound I"

• intmts formed in reactions 2 to 4 have common features in all cytochrome P450s
• similar iron-oxygen states are thought to be important in non-heme oxygen activation

2. Active-Site Structure of P450 Enzymes

• P450 share common overall fold and topology
• Conserved P450 strucural core formed by a four-helix bundle composed of 3 parallel helices D,L, and I and 1 antiparallel helix E.51
• Prosthetic heme group is confined between distal I helix and proximal L helix and boung to adj Cysheme-ligand loop containing P450 signature aa seq. 
• Absolutely conserved Cys is proximal or 5th ligand to heme iron
• This sulfur ligand is thiolate 52
• Prximal Cys forms 2 H bonds with neighbouring backbone amides
• Further interaction with a side chain in some P450s, with Gln in CYP 152A1 or Trp in nitric oxide synthase
• Mutations of these affect reduction potential or catalytic activity and stability of bond between heme iron and its 5th or 6th ligand in NOS
• Long I helix forms a wall of heme pocket and contains signature aa seq (A/G)-Gx(E/D)T which is centred at a kink in middle of helix
• Highly conserved thr preceded by an acidic residue is positioned in active site
• may be involved in catalysis
• SRS predetermine P450 substrate spec
• Point mutations in SRSs affect substrate spec
• SRSs are flesible protein regions
• move on substrate binding in induced fit mech to favour substrate binding

3. Enzymatic Reaction Cycle of Cytochrome P450

• Substrate binds to resting state of low spin (LS) ferric enzyme 
• perturbs water coordinated as 6th ligand of hene iron
• change spin state to high spin (HS) substrate-bound complex
• HS F3+ has more positive reduction potential
• In CYO101 much easier reduced to ferrous state
• In other systems, spin shift is not obligatory part of cycle
• Oxygen binding ---> oxy-450 complex
• last relatively stable intmt in cycle
• Complex reduced
• formation of peroxo-ferric intmt
• formation of its protonated form, hydroperoxo-ferric intmt
• 2nd protonation of distal oxygen atom with heterolysis of O=O bond and formation of Compound I and water
• Oxygenation of substrate to form product complex
• P450 reaction cycle has at least 3 branch points
• multiple side reactions can occur under physiological conditions
• 3 major abortive reaction are

1. autoxidation of oxy-ferrous enzyme with concomitant production of a superoxide anion and return of enz to resting state
2. peroxide shunt. Coordinted peroxide or hydroperoxide anion dissoc from iron forming H2O2,completing unproductive 2-electron reduction of oxygen
3. oxidase uncoupling. Ferry=oxo intmt is oxidised to water instead of oxygenation of substrate. Results in 4-electron reduction of O2 mol. Net formation of 2 mols of water.

• These procseses are uncoupling

3.1. Substrate Binding

• In general substrates for cyt P450 metabolism are hydrophobic
• Substrate binding triggers change of spin state from LS to HS in heme iron
• induce change in reduction potential from ca. -300 to ca. 100 mV more opsitive
• In resting state, or in quilibrium with aerobic media, cyt P450 appear in ferric Fe3+ form
• due to low reduction potential of Fe3+/Fe2+ couple
• -400 to -170mV
• Low redox potential is maintained by prsence of negatively charged proximal thiolate ligand
• same negative shift of reduction potential can be induced in other heme proteins eg myoglobin 85 by replacing proximal His by Cys
• LS-HS thermodynamis equilibria for Fe3+ and Fe2+ states of enz are coupled with 6th ligand binding equilibrium
• Experimentally measured mitpoint potential of heme enz depends on substrate's ability to change heme ligand binding equilibria in ferric and ferrous enz
• In purifies _450 systems, in absence of other stronger ligands, water coordination at 6th distal position can stabilise LS state of ferric iron
• Reduced ferrous cyt P450s are predominantly in HS 5-coordinated state
• water is much weaker ligand for Fe2+ heme
• Difference in ligation state of ferric and ferrous P450 in absence of substrates causes add. stabiliosation of ferric state and lower midpoint potentials of substrate-free cyt P450s.
• Substrate binding causes water mol coordinated to Fe 3+ to be usually displaced
• indicated by shift of spin state of 5-coordinated heme iron to HS
• Loss of 6th ligand of heme iron thermodynamically destabilises ferric state of cyt P450 with respect to Fe2+ state
• midpoint potential of heme shifts to positive values
• Stronger ability of substrate to perturb water ligation of ferric heme, more pronounced resulting positive shift of redox potential
• Same factors affect reduced enz
• In presence of CO or O2 which do not bind ferric iron porphyrins but are strong ligands for Fe2+ heme, reduction potential shifts to more positive values compared to those measured under inert atmosphere.
• Coupling between substrate binding and change in reduction potential is indicated by diff in substrate binding free energy in ferric and ferrous state
• caused by closed thermodynamic cycle.
• Temperature and pH and presence of cosolvents may change parameters of ovserved high-spin-low-spin equilibrium 
• via perturbation of 6th ligand binding 
• change reduction potential of cyt P450
• In most P450 systems ultimate reducing agent for catalytic cycle is NADPH, which has midpoint potential of -320 mV
• reduction potentials of protein's redox partners are roughly in same range
• cyt P450 should be reduced slowly before substrate binds
• Prevents unproductive turnover of enz with waste of NADPH and formatio nof toxic superoxides and peroxides which is rendered more likely due to fast autooxidation of thiolate ligated heme proteins
• Mammalian cytochrome CYP3A4 can bind 2 or 3 substrates mols
• In this system spin shift caused by cooperative substrate binding can serve as allosteric switch
• from slow turnowever at low [S] to faster turnover at high [S]
• Observed cooperativity of product formation may be higher than cooperativity of substrate binding
• Many of cyt P450s have broad spectrum of substrates
• Induced fit model
• Large str rearrangements induced by substrate binding observed by comparing Sray crystal st of cyt P450s 
• Subsrate recognition sites are flexible and procide substrate access to heme
• HEme otherwise buried in protein globule
• In absence of charged and H bonding grps on substrate mols and in the active sites of most P450 enz. such binding mechs stabilise substrate in active centre
• In many cases diff substrate analogues bind tightly to P450 enz because of poor solubility in water
• not because of strong interactions at active site
• Wired substrates bind CYP101 more tightly than natural substrate camphor
• Camphor is tethered to fluorescent reporter grp by hydrophobic links of diff length
• Hydrocarbon tether is dehydrated ---> favourable effect
• Long hydrophobic tail is extended thru binding channel in protein globule up to surface, where fluoresecent grp is partly exposed to solvent
• Same conform changes observed with substrates linked to a Ru complex
• These tethered modified substrates do not dusplace water coordinated to heme iron
• do not shift spin state to high spin, as camphor does
• Spin shift reg mech is sensitive to str of bound substrate or analogue
• enz recognises optimcal substrates this way when there are almost no spec functional grps at active cetnre to control substrate binding spec

3.2. Iron Spin Shift and the Heme Redox Potential

• Some substrates of cyt P450s bind with very high affinity but do not display marked shirt in spin state of ferric he,e
• A crystal str shows no water at 6th ligand position for wired substrate but contains water for another analogue
• Redox potential as Fe3+/Fe2+ equilibrium is perturbed by changes in ligation state
• or changes in ligation strength in course of reduction or oxidation
• In presence of strong ligands for ferric heme redox potential is lower
• Strong ligands for ferrous state will increase redox potential of heme enz
• This concept is valid even without great change in spin state caused by ligand replacement
• equilibrium constants for spin state equilibrium change for diff ligands
• Interaction with other proteins may change heme reduction potential in cyt P450
• In ferric CYP101 studies, spec conform changes of heme, proximal thiolate and key distal pocket residues caused by formation of complex with putidaredoxin
• Interaction with adrenodoxin induces high spin shift in CYP11A1.
• Spin state of heme iron in CYP101 in complex with Pdr coupled with spec change in ESR spectrum of reduced Pdr
• may help function
• Perturb CO stretch band in Fe-CO  complex in CYP 101
• promote e donation to heme iron from axial sulphur ligand of Cys357
• Conform hcange in P450 reductase important in reductase catalysis
• Shift of heme iron spin state from low to high spin
• Ligand concentration or temp can change position of thermodynamic equilibrium with coupled microscopic equilibria
• Redox potential is thermodynamic measure of equilibrium between diff oxidation states
• does not solely determine rate of heme reduction
• Kinetics of reduction depends on spin state change
• reorganisation energy diff involved in changes from a 6-coorfinated to 5-coordinated state
• If reduction is accompanied by changes in coordination state and spin state

          === activation barrier is higher

          ---- rate of reaction is lower

• Eg spni shift alone caused 200-fold increase of reduction rate in substrate-bound wildtpye CYP102 compared to substrate-free mutatns F393A and F393H of same protein
• all 3 enz have similar reduction potentials
• Catalytic turnover is higher in wt enz
• kinetic control of later steps in kinetic cycle, mainly 2nd ET to ferrous-exy complex
• Using resonance raman spectroscopic studies large changes of heme iron reduction potential attributed to conform changes of proponiate and vinyl grps
• Spec conform change caused by substrate binding and formation of complex with ET partners important in reduction kinetics
• Tight binding of steriodal P450 with its redox partner adrenodoxin increase rate of P450 reduction and product formation
• Substrate binding may be rate-limiting step in some P450 systems

          --- low [S]

3.3. Oxygen Complex

• Oxygen binding reduced P450 gives Fe2+-OO complex or Fe3+-OO- complex
• Gross str of oxy-P450 similar to analogous xomplezes in Mb and Hb and heme enzymes, HRP, HO
• In P450 this complex is diamagnetic and EPR silent like Fe2+-OO complex
• only partial electron density transfer from iron to oxygen
• Active proton delivery to bound oxygen or peroxide ligand in P450 mech of oxygen activation
• Autoxidation rate depends strongly on reduction potential in mutants of CYP102
• X-ray crustal str of Fe2+-OO complex of CYP101 was similar to analogous complexes of other heme enz
• O is coordinated in bent end-on mode with angle Fe-O-O 142 degrees
• no steric conflict with bound substrate mol

3.4. Formation of the Peroxo/Hydroperoxo Complex

• Stability of peroxo state Fe3+-OO(H)- is marginal in heme enz
• Prsence of strong proximal ligand (His, Cys or Tyr) and aq sol near neutral pH defines str of most of these complexes as end-on and low-spin state
• Such complexes obtained in reactions with H2O2 with heme enz have low stability
• convert fast to ferryl-oxo-species
• Reactions of peroxide dianion with free Fe3+ porphyrins afford high spin Fe3+-OO2- complexes 

          --- with side-on-bound peroxide and iron displaced out of porphyrin plane towards bound ligand

• To realise this str in heme protein must break proximal ligand bond to iron
• Presence of strong proximal ligand favours low spin state in 6-coordinate Fe3+-OOH- comlexes

          --- important restirction on chem of oxygen activation

          --- characteristic of heme enz

• if system fails to do 2nd protonation at distal oxygen site to promote O-O cleavage

          ---> uncoupling reacion

          --- transition from 5b to resting state of enz

3.5. Peroxoferric Intermediates in Heme

• Enzymes : role of protein transfer
• In heme enz porpyrin and proximal ligand provide heme with 5 coordination sites
• Predent side on of peroxide

         --- common in non-heme metalloenz

         --- very rare in model metalloporphyrin complezes

• Porphyrin donates e for O-O bond cleavage in peroxide ligated to heme iron
• Porphyrin donates 1 e to peroxide ligand

          ----> heterolytic scission of O=O bond

          ----> cation radical forms on pophyrin ring

• Oxyegn activation begins when O2 binds as an axial ligand to Fe2+ heme iron or H2O2 binding Fe3+ heme iron
• Diff between oxidase/ oxygenase pathway an peroxidase/peroxygenase pathway

          --- diff in redox state of oxygen vs peroxide

          --- former pathway needs 2 add reduction steps (1 e reductions by exogenous e donor)

          --- must provide 2 protons delivered to peroxide dianion heme ligand 

• Reultant equivalent of rearranged H2O2, iron-coordinated peroxo-water (Fe-O-OH2) is precursor to heterolytic O-O bond cleavage to form ferryl-oxo porphyrin complex and H2O product
• H bond stabilised network of watre mols is important
• uncoupling of cyt P450 CYP101 mutants with native substrate camphor and wt CYP101 when metabolising other substrates can be conceptualised via operation of distal pocket proton relay system
• Relay composed of water mols stabilised in active centre of enz and 2 residues conserved in P450 systems
• Famous acid-alcohol pair is Asp251-Thr252 in CYP101
• Peroxidase and oxygenase enz use high valent ferryl-oxo pophyrin caton radical as catalyst of oxidative transformation of substrates
• In P450 catalysis O2 binds reduced heme iron
• ferrous-oxygen or ferric-superoxide complex accepts one more e fr protein redox partner
• form peroxo ferric intmt
• protonated to hydroperoxo-ferric intermediate
• contrary to P450 and HO in peroxidases natural formation of active intmt involves HH2O2 as O donor
• H2O2 binds ferrric heme 
• brings 2 e and 2 protons for Compound I generation
• Key step: proton transfer from proximal (closest to iron centre) to distal O atom of bound hydrogen peroxide
• Induce formation of oxo-water by heterolytic splitting of O-O bond
• may be promoted by ET from porphyrin to Fe-O bond
• Water mol and ferryl-ozo porphyrin cation radical are formed
• no other external source of e or protins
• Imidazole side chain of distal His may be intmt catalyst
• accept a proton from proximal oxygen on 1st step
• donates proton to distal oxygen at 2nd step
• pKa of Fe-coordinated Hoo(H) is 3.6-4.0 for HRP and CcP
• Iron-coordinated peroxide is anion at neutral and alkaline pH
• Unstable complex
• Hydroperoxide is weak ligand
• experimentally observed kcat is 10^7 M-1 s-1
• Dissociation rate of HOO(H) may be as high as 10^3-10^4s-1
• Typical for dissoc kinetics of diatomic ligands in heme proteins
• lower than rates of water replacement as a ligand in octahedral porphyrin complexes at room temp
• Protein redox partner reduces heme iron
• P450 enz must catalyse transfer of 2 protons to distal O atom of bound peroxide anion
• thru str arrangement and reulgation of proton relay system
• involves water mols
• stabilised in active centre thru H bonded network interacting with conserved acid-alchohol pair
• Mutating residues in network shows enz kinetics and coupling ratio are sensitive to H bonding properties of sites
• In absence of add catalysis of heterolytic scission thru 2nd protonation of distal O atom  hetereolysis or homlysis of O=O bond is governed by thermodynamic stability of reactant and product
• If iron peroxide complex is mainly in high spin state in non heme metal enz with weak or moderate ligand field

         --- favourable homolysis of O--O bond

         --- spin state of transition state and product state is high spin

• For heterolytic O-O scission with 2nd protonation of leaving O atom and foramtion of water mol

          --- 2nd e supplied to peroxide ligand by porphyrin moiety

          --- porhphyrin cation radical is formed

• determine behaviour of actualy systems in heterolysis/homolysis ratio and product distribution
• Parameters, pH of solvent, ligation of metal, str, redox properties of porphyrin and peroxide may play important roles
• may favour spec catalytic pathway
• by shifting HS-LS equilbrium
• provide protonation of coordinated peroxide
• weak O=O bond
• homolytic decomposition of H2O2 favoured for HS and LS states
• Porphyrin important as e donor for heterolytic scission of O-O bond in coordinated hydroperoxide to form