Hydrogenase enzymes catalyze the reversible oxidation of molecular hydrogen to protons and electrons [H2 ↔ 2H+ + 2e-] .The two most studied classes are Ni-Fe hydrogenases and Fe-only hydrogenases. In general, Ni-Fe hydrogenase enzymes consume molecular hydrogen as a fuel source, and Fe-only hydrogense enzymes produce molecular hydrogen .
Representation of Fe-only Hydrogenase
Crystal structure of the hydrogenase was obtained from a sulfate-reducing microorganism to 1.6 Å resolution (fig 1). Desulfovibrio desulfuricans (DdH) is a 53 kDa protein located in the periplasm . Crystal structure of the enzyme was obtained from Clostridium pasteurianum an anaerobic soil microorganism to 1.8 Å resolution (fig 2). This enzyme, CpI, is a 61 kDa protein found in the cytoplasm . DdH is a dimer with three 4Fe-4S cubanes and a H-cluster (fig 3). CpI is a monomer with three 4Fe-4S cubanes, one 2Fe-2S cluster, and a H-cluster (fig 4).
Figure 1: The DdH Fe-only hydrogenase domain structure. Fe atoms light green, sulfurs in black, nitrogen in blue, oxygen in red, and carbon in gray . fig (2) The CpI Fe-only hydrogenase domain structure. Fe atoms are shown in rust, sulfurs in yellow, nitrogen in blue, carbon in black, oxygen in red; purple indicates the sulfur-bridging moiety of unknown composition . fig (3) The DdH Fe-only hydrogenase contains 3 [4Fe-4S] cubanes and a H-Cluster.fig (4) The CpI Fe-only hydrogenase contains 3 [4Fe-4S] cubanes the 2[4Fe–4S] cubanes, 1 [2Fe-2S] cluster, and a H-Cluster.
Point group symmetry
The overall structure of CpI resembles a mushroom consisting of four domains: the large active site domain forms "cap" and three smaller domains form "stem". The molecular point group for all the structure is C1(fig 4). The "stem" domains bind four iron-sulfur clusters and are termed FS4A-FS4B, FS4C and FS2. FS2 has D2h symmetry(fig 4B). The FS4A-FS4B domain has D2d symmetry(fig 4A). The FS4C domain is placed between the FS2 and FS4A-FS4B domains and consists of two helices linked by a loop that binds a single [Fe4S4] cluster via one His and three Cys residues .
Electronic Structure of the 2Fe Subunit
In order to understand the reactivity of Fe only hydrogenase it is important to know its oxidation state .When the enzyme is isolated in air, we observe an oxidized, inactive state (Hoxair)also known as the EPR silent state. Further reductive treatment results in an oxidized, active state(Hox). The Hox is a low-spin state with a rhombic g = 2.10 signal in EPR. When the Fe-only hydrogenase is fully reduced, another EPR-silent state (Hred) is obtained. DFT calculations show that there are different oxidation states of 2Fe subunit in the system for these three intermediate states (Hox air, Hox and Hred) during catalytic H2 evolution. Calculations are as follows (i) the 2Fe subunit in the Hox air state is most likely a Fe(II)-Fe(II) complex with a hydroxyl (OH-) group bonded at the Fed i.e. Fe(II)-Fe(II)(OH) (ii) a Fe(II)-Fe(I)(vacant) complex is the best candidate for the 2Fe subunit in the Hox state. (iii) the 2Fe subunit in the Hred is a mixture with the major component probably being a protonated Fe(I)-Fe(I) complex. This protonated complex is most likely to mix with its self-arranged form, Fe(II)-Fe(II) hydride.
Few questions arises here.1.Why Fe(II)-Fe(I)(vacant) state is most probable in the system? 2. We know from general inorganic chemistry that Fe(II) and F(III) are stable complexes (due to 3d5 (half occupied) or 3d6 (t2g states occupied) configurations), but Fe(I) is an unstable complex. Then, why the low oxidation states, such as Fe(I)-Fe(I), are stable while the high oxidation states, for example Fe(III)-Fe(III) and Fe(III)-Fe(II), do not exist. I am considering MO theory and back bonding concept to answer above questions which are important for understanding the reactivity of Fe hydrogenase .
Metals with lower oxidation states have a larger electron density. This electron density allows the metal to donate π-electrons from their d orbital to the anitibonding orbital of its ligand, π-back bonding (fig 5). In this situation the π- back bonding stabilizes the bond between the iron and the carbon and destabilizes the bond between the carbon and the oxygen. Also, FTIR
studies show that the oxidation state of Fe(II) must be lower than Fe(I) because we observe a negative shift in the band produced in FTIR spectra. Thus, Fe(II)-Fe(I) oxidation state is most probable for the iron subcluster.
Fig 6 is the interaction diagram between the Fe valence orbitals (3d, 4s, 4p) and the 5σ, 2δorbitals of CO and CN ligands (the interaction between the Fe valence orbitals and the S 2p orbital is omitted here). The energy levels of the 2Fe subunit are shown in the middle. It was found that all the redox states (Fe(II)-Fe(II), Fe(II)- Fe(I), and Fe(I)-Fe(I)) are very similar except for the occupation of a frontier orbital, labeled as eg-2δ. It is unoccupied in Fe(II)-Fe(II), half-occupied in Fe(II)-Fe(I), and fully occupied in Fe(I)-Fe(I) state. Fully filled eg-2δ orbital makes Fe(I)-Fe(I) complex stable. For Fe(III)-Fe(III) and Fe(III)-Fe(II) states, electrons in even stronger bonding orbitals of t2g-2δmust be depleted, which is strongly energetically disfavored. This is the reason why Fe(III)- Fe(III) or Fe(III)-Fe(II) species do not exist .
Reactivity of Fe only hydrogenase
When enzyme is isolated in air it changes its state from Fe(II)-Fe(I)(OH) to Fe(II)-Fe(I)(vacant). Actually, an exchangeable water molecule is bound to the active site labeled “vacant” in the figure. The enzyme then accepts an electron and a proton so that Fe(II)-Fe(I) is oxidized to protonated Fe(I)-Fe(I) Complex.The enzyme changes configuration, and a hydride is transferred into the active site of Fe(II) by reduction. The subsequent addition of an electron and a proton allows for the formation of molecular hydrogen, and the loss of H2 returns the system back to its native active form .
Fe-only Hydrogenase is a very important area of research. Understanding the mechanism of Fe-only Hydrogenase could open the doors for energy sciences development . New developments in the research of Fe-only hydrogenase has peaked interest for use of enzymes in the production of hydrogen.