Mechanism of O2 diffusion and reduction in FeFe hydrogenases

Abstract

FeFe hydrogenases are the most efficient H2 producing enzymes, but inactivation by O2 is an obstacle to using them in biotechnological devices. Here we combine electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations to uncover the molecular mechanism of O2 diffusion within the enzyme and its reactions at the active site. We find that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of the highly reactive OH radical and hydroxylated cysteine, consistent with recent crystallographic evidence. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species at prolonged O2 exposure. These findings should provide important clues for the design of hydrogenase mutants with increased resistance to oxidative damage.

Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments

“…In model (b), we assume that the transport can be taken into account using the Koutecky-Levich approximation, so that: (6) in which j Levich is the so-called Levich current, completely limited by mass-transport (obtained for c 0 = 0) and j enzyme = j (a) is the current given by the system when the transport is infinitely fast (equation (5)). This gives:…”

“…signals resulting from the catalytic oxidation or reduction of the enzyme's substrate at the electrode. The resulting catalytic current, which is proportional to the enzymatic activity, lends itself well to quantitative interpretation: one can monitor its evolution over time to learn about inactivation/activation processes [4][5][6][7], or model its dependence on electrode potential to learn about the steps in the catalytic cycle [8][9][10]. In catalytic PFV, the substrate is consumed at the electrode by the catalytic reaction.…”

O2 sensitivity and H2 production activity of hydrogenases—A review

“…The half‐life of the former in the air‐saturated buffer is in the order of tens of minutes while it is in the order of tens of seconds for the latter. While some [NiFe] hydrogenases are able to reactivate from the O 2 ‐inactivated states, [FeFe] hydrogenases only reactivate from the O 2 ‐bound state to a limited extent (Bingham, ; Kubas et al, ; Orain et al, ). The recovery rate varies depending on the inactivation condition, mainly [H 2 ], duration of O 2 exposure, and redox potential (Kubas et al, ; Vincent et al, ).…”

“…Many [NiFe] hydrogenases are reversibly inhibited by O 2 and can be reactivated after the removal of O 2 (Volbeda et al, ). In contrast, [FeFe] hydrogenases are mostly irreversibly inactivated by even trace amounts of O 2 (Koo, Shiigi, Rohovie, Mehta, & Swartz, ; Kubas et al, ; Shima & Trauer, ).…”

“…These work provided insights into pathways and diffusion channels of O 2 in hydrogenases (Hong & Pachter, ; Kalms et al, ; Mohammadi & Vashisth, ; Wang, Sancho, Best, & Blumberger, ). Their findings provide important clues for the mutagenesis design of hydrogenases to increase the O 2 ‐tolerance level of the enzyme (Kubas et al, ).…”