Iron Hydrogenase 1
Created by Ruth Kihiu
Iron Hydrogenase 1(with a PBD ID of 3c8y) is a protein that falls under a group called Hydrogenases. This particular protein is found in an anaerobic bacterium scientifically named Clostridia pasteurianum (9). This cytoplasmic protein catalyzes the oxidation of dihydrogen into two protons and two electrons and the reverse reaction: the reduction of two protons into a dihydrogen molecule. It is because of these reactions, scientists have studied iron hydrogenase 1. Hydrogen may one day prove to be a valuable and efficient energy source, and the key to synthesizing it lies in the iron hydrogenase 1 (3). The molecular weight of this protein is 63828.13 Daltons and has an isoelectric point (pI) of 6.27.
The function of iron hydrogenase 1 is to break down and synthesize dihydrogen. The synthesizing of hydrogen uses the excess electrons released during the fermentation of pyruvate. The dihydrogen is also used for other metabolic processes that run within the organism (3). This controls excess charges within the cell. The dihydrogen also acts as a fuel source for generate ATP molecules. This protein contains four ligands: 2Fe-2S cluster, glycerol, 2 iron/2 sulfur/3 carbonyl/2 cyanide/water/methylether cluster. The iron containing clusters, also known as ferrodoxins, act as electron donors and acceptors in the uptake and evolution of dihydrogen. The glycerol ligand was part of the protein in the crystallization process. The 2iron/2sulfur/3carbonyl/2cyanide-water methylether cluster make up the center of the activation site. It functions as one unit during the catalytic process (2).
The active site (also known as the H-cluster) has a domain of the iron hydrogenase 1 and it takes up about two-thirds of the entire protein. It consists of residues 210-574. The center of the active site contains the diiron-sulfur linkage with the methylether, cyanide, carbonyl, and water. The two iron atoms (diiron) in the center share a carbon monoxide and are also coordinated to a carbon monoxide and cyanide of their own (6). The carbon monoxide and cyanide molecules help stabilize the diiron cluster at low oxidation states (8). One of diiron atoms is coordinated to the sulfur of a cysteine residue. The other one has an open coordination site. This coordination site is occupied by water when the protein is inactive. The diiron center is also coordinated to a dithiolate ligand. This dithiolate ligand bridges over the center of the active site and contains two sulfurs connected by a molecule. Researchers speculate that this molecule can either be propane or a methylamine. The topic is still up for debate (2). The diiron structure forms an octahedral shape when coordinated to the ligands, but deviates slightly from a true octahedral shape. The sulfur from the cysteine connects the Fe4S4 ligand to center of the ligand. The Fe4S4 is connected in way that creates a cubane structure. Each atom is in a corner of what forms a cube shape. Each iron is bonded to three sulfurs and vice versa for the sulfur atoms (9).
The catalytic process of the protein can go two ways because it deals with the reversible reaction, the uptake of dihydrogen. The concentration of the dihydrogen and the amount of excess electrons in the environment play a part in dictating the direction of the reaction. The first step in the catalytic process is the heterolytic cleavage of dihydrogen to form a hydride and a proton. The electrons and protons are used to synthesize ATP. This is important to metabolic processes because ATP is a biological energy resource. These two molecules bonded to the active site form the intermediates. In order for the catalytic process take place, there needs to be an open coordination site in the active site. The role of the water in center of the coordination site is to act as a weak ligand (2). This makes the protein attractive for displacement to create the hydride intermediate. The placement of other ligands instead of the substrate can inactivate the protein. Carbon monoxide inhibits this protein and the protein cannot reverse this process on its own because carbon monoxide is a viable competitor to the substrate. The carbon monoxide forms a strong bond with the iron. Illuminating the protein at a low temperature (usually below 70 Kelvin) can dissociate the carbon monoxide inhibiting the protein (5). The dihydrogen finds its way to the protein active site by way of a channel. Researchers have discovered a hydrophobic channel (created by the residues of the protein) that facilitates the movement of dihydrogen to the active site. This channel is highly conserved. The ferrodoxins act as proton donors and acceptors during the catalytic process. The dithiolate ligand was considered as a proton donator or acceptor; however researchers have found that this process is energetically unfavorable. Accepting or donating protons can disrupt the diiron structure and affect the function of the protein. A cysteine residue (more specifically Cys-299) could act as a proton donor during the formation of dihydrogen (2).
Several residues are critical to the function of the iron hydrogenase 1. Cys-299, Met-497, Gly-418, Phe-417, Cys-503 are the residues that line the active site (9). The dithiolate ligand is situated between these residues. Hydrogen bonding occurs between the sulfur found in the active site and the water molecules in the protein cavity. The water molecule terminally coordinated to the iron of the diiron cluster is hydrogen bonded to the Cys-299 residue. The Lys-358 is hydrogen bonded to one of the cyanide ligands of the active site. The hydrogen bonding that occurs within the protein is important in the stabilization of the protein and structure. The cysteine that coordinates the diiron cluster to the protein as well as the cubane Fe4S4 structure to the protein is found on the Cys-503 residue. Met-353 also lines the active site. There are a number of hydrophobic residues that surround the diiron cluster. Researchers suggest that these hydrophobic residues protect the cluster from solvent access and also regulate substrate availability. These residues are highly conserved among Fe only hydrogenases. This suggests that the functions of the proteins in this class are similar (3).
Iron Hydrogenase 1 is a Fe-only hydrogenase. There are three types of hydrogenase: Fe-Fe, Ni-Fe, and Ni-Fe-S. The Fe-only hydrogenases have only one type of metal ligand, iron, so it is the only kind without a transition metal. The iron is found at the center of the active site. The structure of iron hydrogenase 1 is crucial to its function. The primary structure of the iron hydrogenase contains 574 residues. Many of the residues are cysteine residues because the sulfur of the cysteine residues coordinates the iron to the protein. Although there are many iron-sulfur linkages throughout the protein, the diiron cluster is the main part of the H cluster. Iron Hydrogenase 1 (CpI) is a monomer (4). The three dimensional structure of this protein is shaped like a mushroom containing four domains and none of these domains overlap. The largest of the domains is the active site, which is located at the “cap” of this mushroom shaped protein. The other three domains make up the “stem” (3).
The secondary structure of protein varies from domain to domain. The active site domain contains two four-stranded beta sheets, each sheet has a number of alpha helices. This domain is also divided into two lobes. Each lobe has one beta sheet with the accompanying number of alpha helices. The left lobe contains the four-stranded parallel beta sheet. Beta sheet strands are from residue 223-230, 263-268, 346-354, and 376-380. The right lobe has the four-stranded mixed beta sheet with the accompanying number of alpha helices. The mixed beta sheet refers to the mixture of parallel and antiparallel beta sheets. These strands run from residues 294-297, 453-461,465-473 and 490-498. The active site is located at the interface of these two lobes. It is where the lobes covalently connect. The two Fe4S4 ligands are located in the domain directly interacting with the active site. The Fe4S4 ligand found closest to the H cluster acts as an electron donor during the reduction of protons to synthesize dihydrogen (3). The other two domains contain other 2Fe-2S ligands. These are found on the cysteine residues but structurally speaking they are not part of the active site. Although these ligands are not found in the active site, the relative closeness of the ferrodoxins to one another suggests that an electron pathway may be formed. The ferrodoxins found close to the surface of the protein or on the surface are the initial electron acceptors. They may be responsible for electron transfer in the oxidation of protons to form dihydrogen. This means that all of the iron atoms found in the protein participate in the catalytic process despite their location in iron hydrogenase 1. The 2Fe-2S ligands that are independent of the active site are coordinated to the protein via Cys-33, Cys-46, Cys-49, and Cys-52. This domain contains two nearly perpendicular mixed beta sheets with one alpha helix. The random coils found in iron hydrogenase 1 connect the alpha helices to the beta sheets in the different domains (3).
One protein that is similar to iron hydrogenase is the Fe- only hydrogenase (with a PDB ID of 1hfe) found in the organism Desulfovibrio desulfuicans. Fe-only hydrogenase, like iron hydrogenase 1 is a hydrogenase that contains only one type of metal ligand. When comparing the primary structure of this protein to that of iron hydrogenase one, they are very close. Using PSI BLAST the primary structure of the main protein (named the query) can be compared to the primary of other proteins to find which ones are most closely related. The E value quantitatively evaluates how similar the sequence of the query is to the sequence of the comparison proteins (also known as subjects). The smaller the E value, the greater the similarity is between the subject and the query. A good E value is one that is below 0.05. The biggest difference between the primary structure of Fe-only hydrogenase and iron hydrogenase 1 is the length of the protein. Fe only hydrogenase contains fewer residues. It has a low E value (1e-90) because the position of the residue relative to the length of the protein is similar in both proteins. Most of the cysteine residues of both of these hydrogenases line up based on the Blast results. This suggests that their iron-sulfur coordination sites are similar in position within each protein. Dali Server is a program that can compare the secondary and tertiary structures of a query to other proteins. The Z score or a comparison protein quantitatively displays the similarity of that protein to the subject. A high Z score signifies that the secondary and tertiary structures are very similar. Fe-only hydrogenase as a high z score (45.3 and rmsd of 2.2). The difference in secondary structure lies between positions 241-255 (it is 363-377 in iron hydrogenase 1). During that interval Fe-only has only random coils but in iron hydrogenase 1those coils are interrupted by beta sheets (1). Some other differences stem from the genetic material used to synthesize the protein. There was a gene splitting event that created the small and large subunit of the Fe-only hydrogenase. Iron hydrogenase 1 is a monomer with non-overlapping domains (2). Other differences are minor changes in the environment around the H-cluster. Fe-only hydrogenase has a cysteine amino acid in the internal pocket (or cavity) of the protein, whereas iron hydrogenase 1 has four water molecules instead. Fe-only hydrogenase and iron hydrogenase 1 serve the same purpose within their respective organisms (3). Their catalytic processes are nearly identical. Both of these proteins have a hydrophobic channel that guides substrates to the active site. The active site of both these proteins and the ligands are the same (2).
Another protein that is similar to the iron hydrogenase 1 is the periplasmic hydrogenase 1 of Clostridium pasteurianum (PDB ID of 1feh). In fact they are identical (E value of zero and Z score of 74 rmsd score of 0.2) (1). Their primary and secondary structure matched up completely when using PSI BLAST and Dali Server respectively. The only difference between these two proteins is their location within the organism. The periplasmic hydrogenase 1 is located in the periplasm of this bacterium. The location is very important to its function. Although the periplasmic hydrogenase 1 is identical to its cytoplasmic counterpart, it creates a gradient when oxidizing. This means that periplasmic catalytic process is coupled with ATP synthesis more often than its cytoplasmic counterpart.
Fe-Fe hydrogenases are different from other hydrogenases in many ways. Although they perform the same tasks, Ni-Fe hydrogenases and Fe-Fe hydrogenases have different catalytic processes. Fe-Fe hydrogenases are engaged in dihydrogen synthesis more often than Ni-Fe hydrogenases because the Fe-Fe counterpart is two orders of magnitude more reactive (4). Unlike in iron hydrogenase 1, Ni-Fe hydrogenases use nickel as their main oxidizing agent for dihydrogen. The center of the Ni-Fe hydrogenase active site is bimetallic and the hydrogen is situated between the metals during catalysis. The bimetallic center is structured a bit differently as well. Although the iron in the bimetallic center has an octahedral shape with its bonds (much like iron hydrogenase 1), the nickel is only bonded to five other atoms. Much like Fe-Fe hydrogenases, Ni-Fe hydrogenases are also inhibited by carbon monoxide (3).
The catalytic processes of iron hydrogenase 1 as well as other hydrogenases have intrigued scientists due to the need to find alternative fuel sources. Hydrogen can prove to be a valuable resource because it is both clean and efficient. It is also a cheap source. Scientists have carefully studied iron hydrogenase 1 in order to attempt replicating a similar process of synthesizing hydrogen. Scientists have also created simple models of the H-cluster to imitate this protein’s function. Fe2(S2C3H6)(CO)6 is one of the simplest models created (7). Current hydrogen vehicles are restricted because of the platinum catalysis used; this proves yet another reason why this protein is so valuable (7). The problem with the protein however is its sensitivity to aerobic environments. Scientists are working to find ways to overcome that obstacle because this enzyme is inhibited by exogenous ligands. Hydroxides and oxygen (in O2 form) will inhibit this protein (2).
Iron Hydrogenase is considered fascinating protein for many reasons. It is one of many types of hydrogenases. It has an effective active site because it can carry out the forward and reverse reactions of the uptake of dihydrogen. This protein utilizes iron as the type of metal for ligand consisting of two iron molecules coordinated to a dithiolate bridge to help synthesize and breakdown dihydrogen. It contains many residues that are crucial to its structure and function. The cysteine residues are important because their sulfur atoms coordinate the iron atoms to the protein. Cys-503 is the residue that holds connects the ligands of the active site to the protein. The residues of iron hydrogenase 1 are highly conserved so they are specific to its function. The analysis of the secondary structure of iron hydrogenase 1 shows that this protein has alpha helices and beta sheets interconnected with random coils. Each of the four domains has its own mix of secondary structures. The fact that the largest domain is the active site domain suggests that the catalysis process is complex. Because of this protein’s ability to synthesize dihydrogen, scientists have tried to replicate its function. Many models have been created, but research is still ongoing.