o binding to mb: spin state
TRANSCRIPT
A.-F. Miller, 2008, pg
O2 binding to Mb: spin state
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Fe2+ + O2 → Fe3+ • O2•-
S =1/2 - 1/2 = 0 : antiferromagnetic coupling
NMR imaging of tissue oxygenation.
S=2
A.-F. Miller, 2008, pg
Comparison of CO, NO, O2
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Fe
OC hν
Fe
OC
Fe
OC
Fe
OC
Upon photolysis:‘geminate recombination’ followed by slower rebinding if recombination is slower than protein dynamics.
A.-F. Miller, 2008, pg
Comparison of CO, NO, O2
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Fe
OC hν
Fe
OC
Fe
OC
Fe
OC
CO geminate recombination in 1 μs, rebinding sameNO 10 ps, rebinding 200 psO2 3 ps, 20 -200 ps, 1 μs (1O2 vs.3O2)
When protein movement is restricted, rebinding is faster!(Protein accommodates departure, stiff protein retains memory of and preference for bound state).
A.-F. Miller, 2008, pg 11
Faster geminate recombination of NO vs. CO (100,000x)
Rate differences α spins of ligands ?: CO has S=0, NO has S=1/2, O2 has S=1
Fe2+ has S=2 (in phorphine)Bound states: Fe-CO S=0, Fe-NO S=1/2 , Fe-O2 S=0
For NO and O2, intermediate spin states are energetically accessible and provide pathways for spin recombination upon binding.
Franzen (2002) PNAS 99:16754
1/2
A.-F. Miller, 2008, pg
Priming of P450 for O2 binding
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LS Fe3+ inert to O2
substrate binds: LS → HS transition and Fe3+ → Fe2+
HS Fe2+binds O2
LS → HS transition is induced by substrate binding.Substrate binding expels a water molecule from Fe3+ coordination. (Amt. of LS character retained α mutant etc.)
A.-F. Miller, 2008, pg
Mechanism of P450
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OH
A.-F. Miller, 2008, pg
Fe3+ spin state change
14 Haines et al (2001) Biochem 40:13456-65.
418 nm α LS394 nm α HS
Kd ≈ 262 nM (vs. 2 μM for palmitic A.)
A.-F. Miller, 2008, pg
Protein conformational
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Cα displacements vs. P450BM3 without substrate:Structural core RMSD is 0.43 Ålid domain residues : lid over substrate access channel
F&G helices close over substrate, along with B’ and N-term. Positions substrate in the AS for rxn to follow.
1JPZ.pdb
A.-F. Miller, 2008, pg 16
Water is expelled from internal channel upon substrate binding. FA side chain is also desolvated. 3 waters remain, 1 in AS.
A.-F. Miller, 2008, pg 17
Helix I conformation change accompanies lid movement (I is fulcrum).In absence of substrate: a water is held at turn between I263 and E267 O’ (A264-T268 N). This kink is conserved. Upon substrate binding, water expelled, C=O•HN Hbd formed between 263 and 267. New water at A264 appears. Helix is now straight.
Sub-bound=pink, H-bds ---no Sub=green, H-bds ---Sub=blue
A.-F. Miller, 2008, pg 18 human Cyt P450 2C9. 1R9O.pdb
Side to which the flavoprotein binds (and delivers electrons from NADPH).
A.-F. Miller, 2008, pg 19 human Cyt P450 2C9. 1R9O.pdb
Active site cavitykink in helix
A.-F. Miller, 2008, pg 20
Kink in helix and conserved Thr side chain proposed to stabilize active site water molecule.
A.-F. Miller, 2008, pg 21
Conformational change: more than just substrate recognition.Decreases in solvent access to active site.Alters Heme reduction potential via change in heme ligation.Opens sixth coordination site for O2 binding.
Binding of O2 requires Fe2+ and open coordination site.
Substrate does not displace aquo directly (no steric clash).F87 could clash with aquo. F87A mutant, LS -> HS transition is complete upon S-binding.Implies that another site becomes more favourable.
Before: Aquo on Fe and H-bond to A264. After: aquo on T268sc and A264. Two sites are mutually exclusive, and OS site is stronger.
A.-F. Miller, 2008, pg
Heme peroxidase
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Cytochrome c peroxidase
ROOH + 2 H+ + 2 e- → ROH + H2O
His
HRP AH2 + H2O2 → A + 2H2O
His
CPO A-H + X- + H+ + H2O2 →AX + 2H2O
Cys-
Catalase 2 H2O2 →2H2O + O2 Tyr-
W•