BACKGROUNDS (1) Figure 1 Active site of CcO

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1 A Cytochrome c Oxidase Model Catalyzes Oxygen to Water Reduction Under Rate-Limiting Electron Flux Collman JP, Devaraj NK, Decréau RA, Yang Y, Yan YL, Ebina W, Eberspacher TA, Chidsey CE. Department of Chemistry, Stanford University, Stanford, CA , USA. jpc@stanford.edu Presentation given by : Vincent Pickenhahn and Suradech Singhanat Catalysis for Energy Production, Master Course, 1st semester 2008/09, EPFL

2 BACKGROUNDS (1) Role of Cytochrome c Oxidase (CcO) -> catalyzes O 2 reduction in the the respiratory electron transport chain without releasing the toxic partially reduced oxygen species (PROS) Mechanism of this enzymatic reduction of O 2 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 Figure 1 Active site of CcO Cu and Tyr -> deliver 1 e - each to bound O 2 Fe from the heme -> gives another 2 e - The oxidized active site is recharged back slowly by ferrrous cytochrom c (cyt c)

3 BACKGROUND (2) Previous works: Models with 2 redox sites, iron heme and copper They can reduce O 2 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

4 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

5 EXPERIMENT AND RESULTS (2) Mimic of e - transfer as rate-limiting step of catalysis Slow SAM S1 : 1-azidohexadecanethiol and hexadecane thiol Fast SAM S2 : azidophenyleneethynylenebenzyl thiol and octathiol Characterization Using conventional electrochemical techniques k 0 : standard e - transfer rate constant between electrode and Fe centre SAM S1 -> 6 ± 0.1 s -1 and SAM S2 -> about 10 4 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)

6 EXPERIMENT AND RESULTS (3) Cyclic Voltammetry Figure 4 A : Slow SAM S1 B : Fast SAM S2 Red : without O 2 Black : with O 2 Absense of O 2 -> redox potentials of Fe and Cu are nearly identical With O 2 -> large irreversible current caused by O 2 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

7 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 disk is set to a potential where O 2 reduction occurs PROS produces during O 2 reduction is pushed away towards the detection Pt ring The ideal four-electron reduction of O 2 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

8 EXPERIMENT AND RESULTS (5) SAM S2 2b (only Fe) -> moderately selective at reducing O 2 to H 2 O 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

9 EXPERIMENT AND RESULTS (6) 2a -> only 3 e - stored at the active site before O 2 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 O 2 selectively by 4 e - under limiting e - transfer rate

10 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 O 2 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 O 2 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

11 THANK YOU FOR YOUR ATTENTION ANY QUESTIONS?