A Cytochrome c Oxidase Model Catalyzes Oxygen to Water Reduction

Under Rate-Limiting Electron FluxCollman JP, Devaraj NK, Decréau RA, Yang Y, Yan YL, Ebina W, Eberspacher TA, Chidsey CE.

Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA. jpc@stanford.edu

Presentation given by : Vincent Pickenhahn and Suradech SinghanatCatalysis for Energy Production, Master Course, 1st semester 2008/09, EPFL

BACKGROUNDS (1)

• Role of Cytochrome c Oxidase (CcO) -> catalyzes O2 reduction in the the respiratory electron transport chain without releasing the toxic partially reduced oxygen species (PROS)

• Mechanism of this enzymatic reduction of O2 is not well understood, just a hypothesis

• It involves the presence of Cu, Fe and Tyrosine residue on the active site of CcO

• Oxygen binds to reduced active site

• Cu and Tyr -> deliver 1 e- each to bound O2

Figure 1 Active site of CcO

• Fe from the heme -> gives another 2 e-

• The oxidized active site is recharged back slowly by ferrrous cytochrom c (cyt c)

BACKGROUND (2)

Previous works:

• Models with 2 redox sites, iron heme and copper

• They can reduce O2 at physiological pH and potential

• But as the models are fixed on graphite electrode, the e- transfer rate is too rapid comparing to cyt c in vivo

This work:

• Use CcO active site models (with Tyr mimic) and its analogs

• Attached them to self-assembled monolayer (SAM) films on gold electrod -> e- can be controlled

• Study the influences of the variation of e- transfer rate on the redox centers. The formation of PROS is also measured

EXPERIMENT AND RESULTS (1)

CcO active site model and its analogs

Electron flux control• Model and analogs covalently attached to SAM-coated gold electrode• e- transfer rate is tuned by varying the length and degree of conjugasion

of the SAM• Attachment -> azide-terminated mixed SAMs and acetylene-bearing

molecules

EXPERIMENT AND RESULTS (2)

Mimic of e- transfer as rate-limiting step of catalysis

• Slow SAM S1 : 1-azidohexadecane-thiol and hexadecane thiol

• Fast SAM S2 : azidophenylene-ethynylenebenzyl thiol and octathiol

Characterization

• Using conventional electrochemical techniques

• k0 : standard e- transfer rate constant between electrode and Fe centre

• SAM S1 -> 6 ± 0.1 s-1 and SAM S2 -> about 104 s-1 (too fast)

• Catalyst coverage is limited -> prevent the interactions between them

Figure 3 Slow SAM S1 with model 1a (left) and Fast SAM S2 with model 1a (right)

EXPERIMENT AND RESULTS (3)

Cyclic Voltammetry

• Absense of O2 -> redox potentials of Fe and Cu are nearly identical

• With O2 -> large irreversible current caused by O2 reduction

• Catalysis takes place at 0.3 V for both slow and fast SAM -> identical to the onset potential observed using native cyt c/CcO complexed

Figure 4

A : Slow SAM S1

B : Fast SAM S2

Red : without O2

Black : with O2

EXPERIMENT AND RESULTS (4)

Selectivity of catalysis under SAM S1 and SAM S2

• Using rotating ring-disk voltammetry technique

• Catalyst-modified SAM-coated gold disk electrode is encircles by a PROS-detecting Pt ring electrode

• Two-electrode assembly is rotated and the gold diskis set to a potential where O2 reduction occurs

• PROS produces during O2 reduction is pushed awaytowards the detection Pt ring

• The ideal four-electron reduction of O2 would not produce any PROS to be detected

• PROS released for the model 1a, its analogs 2a and 2b immobilized on SAM S1 and SAM S2 are measured

EXPERIMENT AND RESULTS (5)

SAM S2

• 2b (only Fe) -> moderately selective at reducing O2 to H2O

• 2a (without phenol) -> 30% decrease of the amount of peroxides released

• 1a -> only slightly improvement on the selectivity observed

• Tyr is not required during the catalysis as e- can be transferred rapidly from the outside of the active site

SAM S1 (in physiological condition)

• 2b -> rapidly degrades and is likely consumed by PROS formation

• 2a -> more stable than 2b, but still remarkable amount of PROS leaked

• 1a -> highly selective, threefold less PROS released

EXPERIMENT AND RESULTS (6)

• 2a -> only 3 e- stored at the active site before O2 binding another one needs to be transfered from the electrode slow transfer -> more PROS

• 1a -> with another e- from phenol, 4 e- required are complete -> Highly selective reduction-> Threefold less PROS

• The selectivity of 1a is nearly identical on slow SAM S1 or fast SAM S2 -> shows its ability to reduce O2 selectively by 4 e- under limiting e- transfer rate

CONCLUSIONS

• When e- transfer limits the turnover rate, the presence of Cu and Tyr mimic reduces sharply the formation of PROS

• CcO catalyst model 1a reduces O2 by 4 e- with 96% selectivity, whereas the native one can do with >99% of selectivity

2 possible explanations for PROS formation

• In CcO, only fully reduced active site can bind to O2 which is not the case for the model 1a -> PROS can be occasionally produced when e- transfer is rate limiting

• The active site of the model and its analogs is exposed directly to the water, whereas CcO is burried in the membrane-> Hydrolytic autooxidation is favored, thus the PROS production is increased (hypothesis)-> This hypothesis is proven by treating SAM with a surfactant which acts as hydrophobic blocking layers

THANK YOU FOR YOUR ATTENTION

ANY QUESTIONS ?