Inorganic Chemistry in Biology: A summary of [NiFe] Hydrogenase
Hydrogenases are a unique class of enzymes that possess the ability to reversibly reduce H+ to H2. One particular enzyme, called nickel-iron hydrogenase ([NiFe] hydrogenase), was originally isolated from the bacterium Desulfovinrio gigas and contains metal and inorganic complexes unusual in most living organisms. While the mechanism of this enzyme is still unknown, its ability to use hydrogen as an energy source is a topic of interest to those looking for an alternative fuel source. Hydrogenases have been found in bacteria, archaea, green algae chloroplast, and subcellular eukaryotic organelles.[1] While different classes of this metalloenzyme exist, [NiFe] hydrogenase is particularly interesting due to the variety of metallic and inorganic complexes that are believed to take part in its mechanism.
Hydrogenases are metalloenzymes that can oxidize molecular hydrogen to protons.[2] An organism containing hydrogenases catalyzes the use of molecular hydrogen as a source of electrons, and reduces compounds such as oxygen, nitrogen, sulfate and carbon dioxide.[3] In the reverse direction, hydrogenases deposit excess electrons onto protons, using them as electron sinks to form molecular hydrogen.(equation 1) [NiFe] hydrogenase is one of several hydrogenases that specifically uses nickel and iron in the active site of the enzyme to catalyze this reaction. A variety of organisms use [NiFe] hydrogenases to produce energy, including both anaerobic and aerobic respirators.
Equation 1
a.) H2 ↔ 2H+ + 2e- 12
b.) H2 + Xoxidized à 2H+ + Xreduced 3 (X= acceptor)
c.) 2H+ + Yreduced à H2 + Yoxidized 3 (Y= donor)
[NiFe] hydrogenase is not specific to either aerobic or anaerobic bacteria. It has been isolated in the bacterium D. gigas and Aquifex aeolicus as well as several prokaryotes. D. gigas is found in anaerobic environments such as sediment or animal gastrointestinal tracts, and uses sulfate rather than oxygen as a final electron accepter in its respiratory cycle.[4] It reduces sulfate to hydrogen sulfide which plays an important role in the ecosystems’ sulfur cycle.4 The hyperthermophilic bacteria, Aquifex Aeolicus, is another bacteria containing [NiFe] hydrogenase, found to exist in an extreme Archaea-like environment, specifically in deep sea vents near volcanos.1 While it is mainly considered anaerobic, A. aeolicus is proposed to be oxygen tolerant, hinting at the wide range of processes that [NiFe] hydrogenases may participate in.1,[5]
Although hydrogenases in the anaerobic respiratory cycle are less well known, their role in aerobic respiration has been characterized in H2 oxidizing prokaryotes. Albracht et al. found [NiFe] hydrogenase to reduce Nicotinamide adenine dinucleotide (NAD) in the cytoplasm of various species. [NiFe] hydrogenase was also discovered to be an integral membrane protein in respiratory chains where O2 is the terminal acceptor..[6]
Due to isoforms and slightly different functions of [NiFe] hydrogenase across species, scientists have found it useful to categorize the enzyme into different groups.1Vignais et al separated [NiFe] hydrogenases into four groups of varying functions:
Group 1 consists of periplasmic [NiFe] hydrogenases. Their task is to remove electrons from molecular hydrogen and send them to membrane-integral cytochromes for electron transport. Periplasmic hydrogenases from sulfate-reducing bacteria comprise a large part of this group.
Group 2 consists of the cytoplasmic [NiFe] hydrogenases. This group consists of hydrogenases involved in hydrogen sensing and uptake of hydrogen in cyanobacteria.
Group 3 consists of hydrogenases that have slightly different structures and are able to bind additional cofactors such as F420, NAD or NADP.
Group 4 consists of the membrane bound hydrogenases involved in hydrogen-producing respiration.3,[7] Brugna-Guiral et al isolated three types of [NiFe] hydrogenases from A. aeolicus, consistent with the perplasmic and cytoplasmic groups of [NiFe] hydrogenases.1
Although huge advances in the study of hydrogenases have occurred over the past 15 years, several ambiguities about the structure and mechanism of this enzyme remain. In 1996, Volbeda et al crystallized the structure of [NiFe] hydrogenase from D. gigas and made important discoveries on the active site orientation. (fig. 2) Of the two metals that exist in the active site only nickel was known prior to their research. Volbeda et al refined the crystal to 2.54Å to unambiguously assign iron as the second metal in the active site complex.[8] With the use of Infrared Spectroscopy, the ligands surrounding the iron center were also elucidated. As the redox state of the enzyme changed, three bands at significantly high frequencies shifted in a concerted fashion.8 Bands at such high frequencies suggested triply bonded molecules that change with the redox state of the enzyme. Two of these molecules are yet to be unambiguously assigned, but they are generally thought to be either CO or CN.8 Volbeda et al originally suggested that the third ligand was NO but Houscroft and Sharpe now claim that IR and mass spectrometry data now show the third ligand to be SO.[9]
Fig. 2
a Image by Alisa Suen
a [NiFe] complex that is the active site of [NiFe] hydrogenase. All sulfur-carbon bonds are part of cysteine amino acid residues, holding the complex in place. Color code: Fe, blue; Mg, green; C, grey; O, red; S, yellow.
Due to the large amount of ligands surrounding both the Fe and Ni center, point groups of the metal complex are more helpful when broken down and generalized. Fe has six immediate ligands making it approximately octahedral and giving it the point group Oh. When the immediate ligands are not generalized, the iron center has C2v symmetry with one C2 principle axis and no center of inversion.(fig. 3) The center also contains two mirror planes, one vertical and one dihedral. Because the iron complex has no center of inversion, the vibrational modes can be both IR and Raman active. Volbeda et al noted that the three non cysteine ligands comprise the three high frequency bands in the IR spectrum and although Raman activity was not recorded, it can be assumed that there would also be three bands in the Raman spectrum.
Fig. 3
a Image by Alisa Suen b Image by Alisa Suen
a Ligands bound to Fe in the NiFe active site. The three unconnected blue atoms on the right hand side of the left picture comprise the sulfurs that bridge the Fe and N. b Generalized structure of Fe with point group Oh. Color code: Fe, blue; C, grey; O, red; S, yellow.
Octahedral molecules can be either high or low spin complexes depending on the magnitude of the change in the d-orbitals. Both CO and CN- are considered sigma electron donors due to high electron density and pi electron acceptors due to empty pi orbitals. The ligand environment surrounding the iron has lead to hypothesis that the Fe(II) center is low spin.9 (fig. 4) The complex is considered low spin because the splitting difference between the pi 1t2g and sigma 2eg orbitals is large. Hence, it is more favorable to fill up the lower energy level than to promote and an electron to a higher level.[10]
Fig. 4
a Image by Alisa Suen
a d-orbital splitting of generalized Fe.
In contrast, the nickel center is surrounded entirely by sulfurs. All but one of these sulfurs comes from surrounding cysteine amino acids holding the two metals together and stabilizing the complex in the active site. Taking into consideration only the immediate ligands on the Ni center, the point group is C4v.(fig. 5) A C4 principle axis can be seen, as well as four vertical mirror planes. Similar to the iron, the nickel has no center of inversion and is both IR and Raman active.
Fig. 5
a Image by Alisa Suen
a Ligands bound to Ni in the NiFe hydrogenase. Generalized structure of Ni with point group C4v. Color code: Ni, green; S, yellow.
Although the mechanism of H2 oxidation is still unknown, there are several substrates besides Ni and Fe in the enzyme that likely play an important role. In the crystal structure of [NiFe] hydrogenase from D. gigas there is an octahedral magnesium held in place by amino acid residues and a water.9,[11] This Mg is relatively close to the NiFe active site and may participate in the catalysis as an electron sink. Close to both the NiFe and Mg centers in an almost linear, alternating combination of iron-sulfur centers. On the edge of the enzyme is the first 4Fe-4S center followed by a 3Fe-4S and another 4Fe-4S. Crystallographic data as well as the known function of iron-sulfur centers in other enzymes, clearly shows this to be the electron transfer pathway in [NiFe] hydrogenase.9 Electrons are thought to be funneled via the iron-sulfur centers to the NiFe active site. The rapid production of protons or reversible H2, catalyzed by this enzyme has made it a prime candidate for use in less-polluting biofuels.
Extensive research is currently being carried out on the industrial production of H2 through biotechnology. Several methods, such as biophotolysis and biocorrosion, are avid producers of useable H2. Biophotolysis was discovered in green algae which uses hydrogenases in photosynthesis to split water into molecular hydrogen and oxygen.[12]
Hydrogenases can also be isolated and with the use of photochemical or electrochemical methods, can create H2.12 Similarly, hydrogenases can generate an electrical current in a biofuel cell by oxidizing hydrogens.12 Alternatively, biocorrosion may eventually be employed to decrease the corrosion of steel. In a proposed model, electrons removed from a cathode are coupled with the production of H2 by [NiFe] hydrogenases, as well as the release of Fe2+ from an anode.12
The perplexity of the metalloenzyme [NiFe] hydrogenase comes from a combined intricacy of both biological and chemical aspects. While numerous phylogenetic, organic and inorganic properties are know about this enzyme, the mechanism, and key to the mass production of what is known to be a useful biofuel, still remains a mystery. New experimental techniques tracking electron channeling will need to be developed to better understand how this enzyme catalyzes the production of protons or reversibly, H2. Communication from all scientific fields will be necessary in order to progress this cutting-edge, alternative energy source.
[1] Brugna-Gurial, M.; Tron, P.; Nitschke, W.; Stetter, K. O.; Burlat, B.; Guigliarelli, B.; Bruschi, M.; Giudici-Orticoni, M. T. “[NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics.” PubMed. 2003, 145-57.
[2] Adams, M.W.W. and Stiefel, E.I. (1998). "Biological hydrogen production: Not so elementary".Science282: 1842–1843.
[3] Vignais, P.M., Billoud, B. and Meyer, J. (2001). "Classification and phylogeny of hydrogenases".FEMS Microbiol. Rev.25: 455–501.
[4] Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. "Microbial Metabolism."Microbiology: an Introduction. 10th ed. San Francisco: Pearson Benjamin Cummings, 2007. 132+. Print.
[5] Pandelia, M. E.; Fourmond, V.; Tron-Infossi, P.; Lojou, E.; Bertrand, P.; Leger, C.; Giudici-Orticoni, M. T.; Lubitz, W. “Membrane-bound hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus: enzyme activation, redox intermediates and oxygen tolerance.” J. Am. Chem. Soc. 2010, 132, 6991-7004.
[6] Albracht S.J.P. “Intimate relationships of the large and the small subunits of all nickel hydrogenases with two nuclear encoded subunits of mitochondrial NADH: ubiquinone oxidoreductase.” Biochem Biophys Acta. 1993, 1144, 221-224.
[7] Wu, L. F.; Mandrand, M. A. “Mircobial hydrogenases: primary structure, classification, signatures and phylogeny.” FEMS Mircobial Rev. 1993, 104, 243-270.
[8] Volbeda, A.; Garcin, E.; Piras, C.; de Lacey, A.L.; Fernandez, V.M.; Hatchikian, E.C.; Frey, M.; Fontecilla-Camps, J.C. “Structure of the [NiFe] hydrogenase active site: evidence for biologically uncommon Fe lignads.” J. Am. Chem. Soc. 1996, 118, 12989-12996.
[9] Housecroft, Catherine E., and A. G. Sharpe. "Biological Redox Processes."Inorganic Chemistry. 3rd ed. Harlow, England: Pearson Prentice Hall, 2008. 982-84. Print.
[10] Berben, L. Chemistry 124, Inorganic Fundamentals. May 10, 2010. Lecture 19.
[11] 2FRV -PDB structure of [NiFe]-hydrogenase fromDesulfovibrio gigas
[12] Mertens, R.; Liese, A. “Biotechnological applications of hydrogenases.” Current Opinion in Biotechnology. 2004, 15, 343-348.