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Biological H2 production from [FeFe]-hydrogenase, found in photosynthetic microorganisms,

is an attractive alternative source of renewable energy in future [22, 23, 24]. Among such microorganisms, green algae and cyanobacteria are potential sources of H2 production be-

cause they have a maximum solar to hydrogen (STH) conversion efficiency [11, 25, 26]. The hydrogenase family is broadly classified into [NiFe]-hydrogenase and [FeFe]-hydrogenase, based upon the metal-cluster present in the active-site [27, 28]. In comparison to [NiFe]- hydrogenase, [FeFe]-hydrogenase is primarily responsible for H2 production, but is signif-

icantly inhibited by O2 [29, 30]. In this work, I have focused on CpI [FeFe]-hydrogenase

from Clostridium pasteurianum. Fig. 1.2 shows the crystal structure (PDB code 1FEH) of the CpI [FeFe]-hydrogenase containing 574 amino-acid residues, and six metal clusters: four [Fe4S4] metal clusters, three of which are ligated to four cysteines, distal metal cluster that

is ligated to three cysteines and one histidine, and one surface cluster [Fe2S2] that is ligated

to four cysteines.

The central metal cluster, which is known as the H-cluster, consists of [2Fe]-subclusters with ligation to carbonyl and cyanide groups [31]. Hydrogen production in this enzyme takes place at the H-cluster buried inside the protein [32]. In [FeFe]-hydrogenase, the H-cluster is a complex structure consisting of Fe2(CO)3(CN)2(dithiomethylamine), which is covalently

Figure 1.2: Locations of metal clusters in [FeFe]-hydrogenase are highlighted. The protein- backbone is shown as ribbons, and metallic clusters are shown as space-filling. The active-site domain of the protein is shown in blue ribbons, and the non-active-site domain is shown in red ribbons.

bound to an accessory metal cluster [Fe4S4] [33, 34, 35]. The side of the protein that harbors

the H-cluster (blue ribbons in Fig. 1.2), is designated as the active-site domain, and the side of the protein that harbors other metal clusters, is designated as the non-active-site domain (red ribbons in Fig. 1.2).

A major obstacle to achieving the goal of sustained H2 production is the inactivation of this

hydrogenase by oxygen [25, 26]. Although both NiFe and FeFe hydrogenases are sensitive to inactivation by O2, [NiFe]-hydrogenases capable of naturally resisting the inactivation

by O2 with different metallic clusters were recently found [36, 37]. Recently, success has

also been achieved in improving the O2 sensitivity of [NiFe]-hydrogenase [38, 39, 40], but

no similar progress has been made on H2-producing [FeFe]-hydrogenase. It is thought that

the inactivation of [FeFe]-hydrogenase by O2 consists of diffusion of O2 from the solvent into

Although the exact mechanism of inactivation of the H-cluster after binding of O2 is not

well understood, X-ray absorption measurements show that binding of O2 to the distal Fe

(Fed) in the H-cluster can subsequently damage the [Fe4S4] accessory cluster next to the H-

cluster [43, 44, 45, 46, 47]. It has been proposed that possible formation of reactive oxygen upon O2 binding to Fedof the H-cluster and its diffusion toward the [Fe4S4] accessory-cluster

damages this cluster (cluster 1 in Fig. 1.2) [43, 48]. However, Swanson et al. [49] describe a rather complex mechanism for degradation of the H-cluster, including the formation of a reversible state before its full degradation. Aside from the H-cluster, Liebgott et al. [50] have shown that by altering the structure of [NiFe]-hydrogenase using a site-directed mutagenesis approach, the diffusion rate of O2can be slowed by orders of magnitude, thereby

slowing the inactivation rate. This is accomplished by replacing the amino acids along known gas diffusion channels with bulky amino acids, likely blocking the possible O2 diffusion

path within the protein [50]. Similarly, Nienhaus et al. [51, 52] show that CO diffusion in myoglobin is altered by mutations of amino acids along CO-diffusion paths.

The presence of gas channels in [FeFe]-hydrogenase for O2 and H2 has been investigated both

computationally and experimentally. The existence of two main gas channels in the active- site domain of the [FeFe]-hydrogenase has been proposed [5, 11, 53, 25]. Kubas et al. [10] investigated the rate of O2 diffusion along these two main channels [10]. A major limitation

of these investigations has been a narrow understanding of O2 diffusion pathways within

the other parts of [FeFe]-hydrogenase, and interactions of these diffusion channels with each other. Moreover, computational studies for gaining insights into ligand diffusion mechanisms, such as O2 diffusion in [FeFe]-hydrogenase, is still an active area of research [54]. In fact,

Bingham et al. [55] have reported that the mutations in an area away from the two main gas channels affect O2diffusion noticeably, indicating the potential presence of other gas channels

in [FeFe]-hydrogenase or an allosteric communication between O2 binding sites. Although,

both Lautier et al.’s [6] and Ghirardi et al.’s [11] mutagenesis investigations suggest that O2

for O2 within the protein was highlighted [6]. Indeed, a network of interconnected diffusion

pathways have been found for ligand diffusion in various proteins [56, 57, 58], clearly showing the presence of key residues or reaction sites along the predicted pathways, and elucidating the interconnected nature of diffusion pathways of ligands in proteins. Therefore, a better understanding of gas diffusion in [FeFe]-hydrogenase is needed to develop strategies for its functional modification. Thus, a detailed mapping of diffusion pathways of CO and O2 in the FeFe-hydrogenase is not only needed for resolving these questions, but also for developing approaches to enhance the tolerance of the FeFe-hydrogenase to inhibitory gases.