1. cv_reac_5c2.ppt / 040608 * : S=1/2 active Ni a -S 1931 Ni a -C* 1950 Ni a -SR 1936 Activation/ Inactivation H2H2 H2H2 inactive Ni u * 1945 Ni u -S 1948 Ni r * 1943 Ni r -S 1931 secs What leads to oxidative inactivation, and how is it reversed ? ‘Unready’ Ni-A ‘Ready’ Ni-B ? Ni(III) Ni(II) v. slow

2. Hydrogenase adsorbed on PGE electrode, pH 9.0, H 2 at approx 1 atm, Scan rate 0.3 mV/s. electrode rotating at 1500 rpm, temp 45 o C. Jones et al. JACS 125, 8505 (2003) Potential / V vs SHE H 2 catalytic oxidation current InactiveActive Oxidative cycles at slow scan rates reveal the anaerobic interconversion between active and the ‘Ready’ state of NiFe Hydrogenase. Anne Jones

3. Potential / V vs SHE H 2 oxidation activity Inactivate Activate

4. time/s Study kinetics of inactivation by applying steps to high -potential Rates of oxidative inactivation are independent of potential (electrodic driving force) (results at pH 8.8) Jones et al JACS, 2003 potential steps 

5. Study kinetics of activation by applying steps to low -potential Enzyme is easily reactivated Rate increases as potential is lowered (i.e. as driving force is raised) Jones et al JACS, 2003 semi-log plots increasing driving force (more negative potentials)

6. ‘unready’ ‘ready’ Ni(III) Ni(II) E C Ni III –OH Fe Ni II –OH FeNi II (H 2 O) Fe e-e- H+H+

7. ADDING O 2 at -0.158 V vs. SHE, pH 9, 45 o C. 0.3 mV s -1. O 2 attacks site directly,and enzyme inactivated much faster than observed for anaerobic reaction. But most activity is regained rapidly when the scan direction is reversed, at the same potential as for the anaerobic inactivation. inject O 2 i (  A) Potential /V vs SHE -0.6-0.4-0.20.40.20.0 1.6 1.2 0.8 0.4 0.0 Lamle et al JACS 2004

8. 0500100015002000 0 10 20 30 40 current (  A) Time (s) Fast phase of reactivation (READY) Slow phase of reactivation (UNREADY) -88 mV step to -88 mV to reactivate step to 242 mV add O 2 then purge headgas with H 2 (iii) The O 2 injection and potential-step sequence experiment; pH 6, 45 o C O 2 added to enzyme while it turns over H 2

9. O 2 was injected at 42 mV / H 2 O 2 was injected at 42 mV under N 2 O 2 was injected at 242 mV under N 2 Normalized i 0.50 1.00 0.75 0.25 0.00 Time (s) 200015001000500 O 2 was injected at 217 mV under H 2 Injection O 2 under Ar or N 2 generates more ‘Unready’ than injecting O 2 under H 2 and there is marked dependence on electrode potential Activation plot for slow phase  H 88 kJ/mol t 1/2 = 280 sec at 45 o C Lamle et al JACS 2004

10. E vs SHE % Unready Under N 2 Under H 2 0 20 40 60 80 100 -0.050.00.050.10.150.20.250.30.35-0.1 Unready is formed when the active enzyme reacts with O 2 and there are not many electrons available ! + CO before O 2 pH 9 Lamle et al JACS 2004

11. Hypothesis... When active enzyme is drained of electrons, exposure to O 2 produces ‘Unready’ state in which O 2 is not fully reduced. But if electrons are readily available, O 2 is reduced to ‘water’ and ‘Ready’ state is formed.

12. Ni II Fe II + O 2 + 4e - + 3H + Ni III -O-Fe II + H 2 O Ni II Fe II + O 2 + 2e - + H + Ni III Fe II H ‘Ready’ (Ni-B) ‘Unready’ (Ni-A) (Blocked) [O] H2OH2O+ trapped O-atom species The difference between Ni-A (Unready) and Ni-B (Ready)

13. Ni-B (‘Ready’) nearly pure state Ni-A or Ni-SU (‘Unready’) New crystallographic refinement leads to re-interpretation of the earlier structure of enzyme crystallised mainly in the ‘Unready’ state (Fontecilla-Camps).

14. Normalized current Time / sec -8 mV -158 mV -58 mV -33 mV -78 mV Reductive activation of Unready. As reductive step potential is raised, rate of activation slows down. Excellent first-order traces in all cases

15. Rate / s -1 Potential / V vs SHE pH 6.0 pH 7.0 Plot reveals sharp potential dependence of rate of activation of Unready state (5-100% H 2 ) limiting rate reached at -100 mV (pH 6)

16. Does Unready form of hydrogenase activate without H 2 ? current

17. Measure extent of ‘activation’ under N 2 as function of potential Inject O 2 -228 mV -158 mV 242 mV, N 2 -228 mV/ -158 mV H2H2 -558 mV, N 2 H 2 oxidation current time / s

18. In absence of H 2, reduction of Unready proceeds to a position of equilibrium Lamle et al. in preparation