O binding to Mb: spin state

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


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.


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


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.)


Mechanism of P450

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OH


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.)


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


Water is expelled from internal channel upon substrate binding. FA side chain is also desolvated. 3 waters remain, 1 in AS.


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


Kink in helix and conserved Thr side chain proposed to stabilize active site water molecule.


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.


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•

O binding to Mb: spin state

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