How an oxygen-tolerant hydrogenase protects itself from oxygen

09-02-2012

In the future, biocatalysts will become ever more important in processes aimed at providing alternative, and greener, fuel sources. However, the production of enzymatic fuel cells involving hydrogenases is currently hampered because the activity of these enzymes is inhibited by the presence of oxygen. The crystal structure of an oxygen-tolerant hydrogenase has revealed the mechanism used by some of these enzymes to protect themselves from oxygen, an observation that should pave the way for the expanded use of hydrogenases in many biotechnological developments.

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Hydrogenases - biocatalysts that convert molecular hydrogen into protons and electrons - will be crucial in the future development of enzymatic fuel cells. Unfortunately, the biotechnological application of hydrogenases is currently limited as the activity of most such enzymes is severely attenuated, or even destroyed, in the presence of oxygen.  Oxygen-tolerant hydrogenases do exist in Nature and have been used in the light-dependent production of hydrogen by coupling them to the photosynthetic apparatus of cyanobacteria [1].  Determining mechanisms of oxygen-tolerance is thus very important if enzymatic fuel cells are to become an alternative source of fuel. The crystal structure, solved using diffraction data collected at ESRF beamline ID14-4 and at BESSY II, of a hydrogenase (MBH, membrane-bound hydrogenase from Ralstonia eutropha H16) that is active even at high concentrations of O2 has revealed how it 'protects' itself from oxygen.

 

A ribbon representation of the three-dimensional structure of MBH.

Figure 1. A ribbon representation of the three-dimensional structure of MBH. The [NiFe] catalytic centre and the three Fe-S clusters involved in electron transport are shown as spheres. The spatial arrangement of the Fe-S clusters is also shown, as is a depiction of the cellular localisation of MBH. (Credit: J. Fritsch et al., Nature 479, 249–252 (2011), reprinted by permission from Macmillan Publishers Ltd, copyright 2011.)

The crystal structure of MBH shows that three iron-sulphur clusters are involved in the electron transport required for enzymatic activity (Figure 1). The determining factor in the oxygen tolerance of MBH is the presence, close to the [NiFe] active site, of a novel [4Fe-3S] cluster coordinated by the sulphur atoms of six cystein residues, two of which are found exclusively in oxygen-tolerant hydrogenases (Figure 2). The novel cluster, which adopts an open, non-cuboidal conformation with enlarged Fe–Fe distances, is able to adopt three redox states at physiological conditions and is proposed to act as a switch which serves either as an electron acceptor upon H2 oxidation or as an electron donor which, upon oxygen attack, delivers the electrons required for the reduction of O2 to water, thus freeing up the active site and allowing hydrogenase activity to continue.

The non-cuboidal structure and coordination sphere of the novel [4Fe-3S] cluster.

Figure 2. The non-cuboidal structure and coordination sphere of the novel [4Fe-3S] cluster that determines the oxygen tolerance of MBH. (Credit: J. Fritsch et al., Nature 479, 249–252 (2011), reprinted by permission from Macmillan Publishers Ltd, copyright 2011.)

The oxygen tolerance of hydrogenases results in a continuous production at the active site of water which must be continuously removed from the protein core.  In addition to the novel [4Fe-3S] cluster, the crystal structure also revealed the presence of cavities, filled with water, connecting the active site of MBH with the surface of the protein (Figure 3).  The disposition of the amino acids lining these cavities, which are absent in the structures of oxygen-sensitive [NiFe]-hydrogenases, suggests a pathway for the controlled movement of water molecules away from the active site. These cavities are clearly also crucial to the oxygen tolerance of MBH.

The translocation of water molecules away from the active site of MBH.

Figure 3. The translocation of water molecules away from the active site of MBH.  The gas tunnel funnelling hydrogen and oxygen towards the catalytic centre is shown as a grey surface, the water (red spheres) filled cavities observed in the crystal structure are shown as blue surfaces. (Credit: J. Fritsch et al., Nature 479, 249–252 (2011), reprinted by permission from Macmillan Publishers Ltd, copyright 2011.)

The elucidation of the mechanism of the oxygen tolerance of MBH should lead to the increased use of hydrogenases in biotechnological applications such as light-driven hydrogen production or in biological fuel cells for power generation. Moreover, it is likely that the unearthing of the novel [4Fe-3S] cluster with its exceptional redox properties will result in the rational design of more efficient oxygen tolerant hydrogenases, thus further expanding their use in biotechnology.

 

Principal publication and authors
The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre, J. Fritsch (a), P. Scheerer (b), S. Frielingsdorf (a), S. Kroschinsky (b), B. Friedrich (a), O. Lenz (a) & C.M.T. Spahn (b,c), Nature 479, 249–252 (2011).
(a) Mikrobiologie, Institut für Biologie, Humboldt-Universität zu Berlin (Germany)
(b) Institut für Medizinische Physik und Biophysik (CC2), Charité–Universitätsmedizin Berlin (Germany)
(c) Zentrum für Biophysik und Bioinformatik, Humboldt-Universität zu Berlin (Germany)

 

References
[1] B. Friedrich, J. Fritsch & O. Lenz, Curr. Opin. Biotechnol. 22, 358–364 (2011).

 

Article written by G. Leonard, ESRF.

 

Top image: cellular localisation of MBH.