Absorbing oxygen: Understanding enzymes

Undoing enzymes

New clues as to why oxygen becomes the undoing of hydrogenase enzymes has emerged from X-ray absorption spectroscopy studies carried out at the Swiss Light Source.

Enzymes used for the production of hydrogen are very sensitive to the presence of oxygen. Now, researchers in Germany have investigated the time course of the processes involved to reveal what deactivates the enzyme's iron centre.

"Hydrogenases could be extremely significant for the production of hydrogen with the help of biological or chemical catalysts," explains Camilla Lambertz of the Department of Plant Biochemistry at Ruhr-Universitaet-Bochum, "Their extreme sensitivity to oxygen is however a major problem." If such enzymes are to be commercially viable in the production of renewable hydrogen for our future carbon-reduced fuel economy then researchers such as Lambertz and her colleagues, Jens Noth, Martin Winkler and group leader Thomas Happe, need to learn more about them and to find ways to develop more robust versions of the natural enzymes.

Fundamentally hydrogen

Fundamentally, hydrogenase enzymes simply convert protons and electrons into molecular hydrogen. The team are investigating [FeFe]-hydrogenases as a specific example of hydrogen-generating enzymes; they focused on HydA1 from the green alga Chlamydomonas reinhardtii. They explain that these enzymes can produce large quantities of hydrogen via their active H-cluster, which consists of a di-iron and four-iron sub-cluster and associated ligands. The team has collaborated with Michael Haumann's group (with Nils Leidel, Kajsa Havelius and Petko Chernev) in the Institute for Experimental Physics at Freie Universität Berlin, to find out how oxygen molecules bind to this di-iron centre to deactivate another enzyme component containing four other iron atoms. The team has now revealed the details of the inactivation process for the first time using X-ray absorption spectroscopy. The study was carried out using the synchrotron radiation source Swiss Light Source. The study involved exposing enzyme samples to oxygen for different lengths of time, seconds, minutes and hours and deep-freezing them in liquid nitrogen.

The researchers were then able to use the spectroscopic data thus obtained to develop a model for the three-phase inactivation process that apparently occurs in hydrogenase enzymes.

The team describes the distinct phases of the process thus: "The first phase [less than 4 seconds] is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2FeH), and oxidation of one Fe ion. The second phase (15 s) causes a ~50% decrease of the number of ~2.7 Å Fe-Fe distances in the [4Fe4S] sub-cluster and the oxidation of one more Fe ion. The final phase (1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation." In other words, their model suggests that initially an oxygen molecule binds to the di-iron centre of the hydrogenase, which generates a highly oxidising species, which then attacks and modifies the four-iron centre. In the final stage additional oxygen molecules bind and lead to the disintegration of the complex.

Stepwise disintegration

"The entire process thus consists of a number of consecutive reactions that are separated in time," explains Lambertz. "The velocity of the entire process is possibly dependent on the phase during which the aggressive oxygen species moves from the di-iron to the four-iron centre." The team is currently designing follow up experiments in order to try and verify this hypothesis.

Improved understanding might allow biotechnologists to engineer alternative enzyme structures that might be just as active in hydrogen generation for a commercial fermentation process but that would not simply disintegrate in the presence of molecular oxygen. "Such progress may facilitate their use in devices for sunlight- driven fuel production, in which water oxidizing catalysts, producing O₂ as a by-product, generate electrons for coupled H₂ formation at the H-cluster," the team concludes.

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