How oxygen attacks [FeFe] hydrogenases from photosynthetic organisms

Abstract

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.EXAFS ͉ H-cluster ͉ protein film electrochemistry ͉ biological hydrogen production ͉ green algae

Electrocatalytic mechanism of reversible hydrogen cycling by enzymes and distinctions between the major classes of hydrogenases

“…In the case of CaHydA, for which negligible inactivation occurs at high potential, it is clear that the oxidation current reaches an almost flat limiting value, consistent with H 2 oxidation becoming controlled by a chemical rather than an electrochemical step (under 100% H 2 the oxidation current is independent of rotation rate above 2,000 rpm and so is not controlled by mass transport of H 2 to the electrode). The [FeFe]-hydrogenase CrHydA1 consistently shows a small inflection in the catalytic current at potentials slightly above and below E 2H þ ∕H 2 (30). The H þ reduction activity of both [FeFe]-hydrogenases increases strongly as the electrode potential becomes more negative.…”

Photoautotrophic hydrogen production by eukaryotic microalgae under aerobic conditions

“…Relatively higher H 2 ase activity was observed under lower initial O 2 conditions (5-10%) compared with the higher O 2 environment (atmospheric O 2 ), but no apparent correlation was observed between the specific H 2 ase activity and the microalgal biomass amount as the activity significantly varied for similar biomass when the inorganic carbon concentration was fixed at 10% (Figs 1f and 4). H 2 ase is extremely sensitive to oxygen, and its activity is inhibited even on exposure to low concentrations of oxygen 19 . However, in the present study, H 2 ase activity at partial pressure of oxygen (5-21%) consistently indicated that the microalgae could perform hydrogen production and/or H 2 ase synthesis, even in 21% oxygen (Figs 2 and 4 and Supplementary Fig.…”

Investigation of the Ultrafast Dynamics Occurring during Unsensitized Photocatalytic H2 Evolution by an [FeFe]-Hydrogenase Subsite Analogue

“…The enzymes themselves are extremely effective catalysts of the reduction of protons to dihydrogen in vivo but are not technologically viable for several reasons, not least their sensitivity to oxygen. 1 In recent years, it has been shown that many synthetic compounds based loosely on the active site structure of these enzymes also have the ability to produce hydrogen. [2][3][4][5][6][7][8][9] These iron-or nickel-based mimics are particularly attractive as they offer the possibility of replacing the platinum-based systems currently in technological use with more economically-viable materials.…”