Bioinorganic chemistry

Enzymes activated by synthetic components

Synthetic analogues of the catalytic subsite of the hydrogen-producing enzyme HydA1 have been disappointingly inactive. The incorporation of such analogues into the enzyme's active site reveals the requirements for activity. See Letter p.66

As our knowledge of biosynthetic pathways evolves, experiments that interfere at specific points in these molecular assembly lines may be judiciously designed. On page 66 of this issue, Berggren et al.1 report just such a strategy in their study of a [FeFe]-hydrogenase enzyme called HydA1, which mediates the remarkably efficient production of hydrogen gas from water-derived hydrogen ions2,3,4,5,6,7,8. The authors' findings increase our understanding of how the enzyme's active site is constructedFootnote 1.

Buried deep within HydA1, the enzyme's active site consists of a [4Fe-4S] cluster (a group of four iron and four sulphur atoms), which serves as a storage and conduit unit for electrons, and a [2Fe] subsite that is the real engine of the catalyst4. The [2Fe] subsite is actually a small molecule in which two iron atoms are bound by diatomic ligand molecules (carbon monoxide and cyanide) and connected by a unique dithiolate bridge, SCH2XCH2S, where the identity of X has been contentious, but could be carbon (CH2), oxygen or nitrogen (NH). The subsite is attached to the protein only at the embedded [4Fe-4S] cluster, through a bridge formed by the sulphur atom from a cysteine amino-acid residue.

Organometallic chemists have made synthetic analogues of the subsite that resemble its structure, but these exhibit low catalytic activity for the hydrogen-forming reaction in the absence of the protein. A fundamental question is whether these analogues of a small molecule can be recognized at the appropriate point in the biological assembly of the active site of HydA1, and so be inserted into that site. If incorporated into the incomplete protein, would synthetic [2Fe] units be catalytically active? And could the insertion of synthetic analogues into the site be used as a technique to interrogate why their activity is low? More specifically, could this approach be used to identify the elusive bridgehead X of the dithiolate group?

Although a lot is known about the events that control the generation and combination of the components of the [2Fe] subsite, and about the maturase proteins involved6, much remains to be clarified. Nevertheless, it is widely accepted that the [2Fe] subsite is built on a scaffold provided by the 'apo' (incomplete) form of an isolable protein known as HydF (ref. 7). Once the [2Fe] unit is formed, the resulting 'holo' (complete) form of HydF serves as a delivery agent, shuttling its cargo to apo-HydA1, where the required [4Fe-4S] cluster resides at the end of a deep cavity3. On acceptance of the [2Fe] subunit, HydA1 matures: the channel that provided access for the subunit collapses, generating the complete hydrogenase enzyme in which the [4Fe-4S] cluster and the [2Fe] subunit are fully encapsulated. It has been postulated that cavity collapse causes one carbon monoxide ligand to be lost from the [2Fe] subunit and another to shift into a bridging position between the two irons. This would change the [2Fe] subunit from a symmetrical structure (akin to the structures of its synthetic analogues) to a 'rotated' isomer that is catalytically active9.

Berggren et al. prepared apo-HydF from the bacterium Thermotoga maritima by overexpressing it in Escherichia coli bacteria, and then added it to three synthetic analogues of the [2Fe] subunit. The three analogues differed only in the bridgehead atom X, which was carbon, nitrogen or oxygen. Fortunately, the diatomic ligands in the analogues can be easily detected using infrared spectroscopy. This enabled the researchers to track the artificial subunits as they passed from solution into HydF, and eventually into HydA1. Even better, the spectroscopic signatures of synthetic and natural [2Fe] subsites are highly sensitive to changes in their environment. This allowed Berggren and colleagues to confirm that HydF proteins did indeed bind to the synthetic [2Fe] subunits, and that the chemical environment of the diatomic ligands was similar to that found in the isolated, natural form of the protein.

The authors used another spectroscopic technique, electron paramagnetic resonance, to determine how the synthetic [2Fe] subsites are attached to the [4Fe-4S] clusters. This revealed an unexpected role for a cyanide ligand in the subsites. Cyanide ligands have long been known to aggregate metals, as in the widely used pigment Prussian blue. Berggren et al. suggest that this ability allows a cyanide ligand to act as a temporary connection between an iron atom in the subsite and one of those in a [4Fe-4S] cluster of apo-HydF (Fig. 1). As HydF transfers its package to apo-HydA1, a cysteine sulphur atom binds the [2Fe] site to form mature HydA1, the cyanide detaches from the HydF cluster and a carbon monoxide ligand is lost from the subsite.

Figure 1: Assembly line interrupted.

Berggren et al.1 have bypassed much of the biosynthesis of the HydA1 enzyme, to introduce synthetic analogues of the enzyme's [2Fe] subsite. They observed that the analogues first become incorporated into the apo (incomplete) HydF protein by binding to its [4Fe-4S] cluster, and propose that a cyanide ligand acts as a bridge between iron atoms in the cluster and the subsite analogue. This temporary bridge is used during the transfer of the [2Fe] unit to a similar [4Fe-4S] cluster in apo-HydA1: a sulphur atom (green) from a cysteine amino-acid residue in HydA1 latches onto the [2Fe] cargo, releasing a carbon monoxide (CO) ligand from the subsite and yielding the mature form of HydA1.

So, are the semi-synthetic enzymes functional? To answer this question, Berggren and co-workers combined apo-HydA1 with one of the following: an empty scaffold (apo-HydF); HydF bound to the naturally occurring [2Fe] subsite; or HydF bound to each of the artificial [2Fe] subsites. The authors found that no hydrogen gas was produced in reactions using apo-HydF, or for scaffolds bound to artificial [2Fe] subsites in which the bridgehead atom X was carbon or oxygen. However, when they tested the [2Fe] subsite that had a nitrogen bridgehead, they observed vigorous hydrogen-gas production, comparable to that of the naturally occurring enzyme. Notably, under the same assay conditions and in the absence of apo-HydA1, HydF loaded with the nitrogen-bridged subsite was inactive.

The researchers' findings beg the question, can apo-HydA1 be loaded with a synthetic [2Fe] analogue in the absence of HydF? Something similar has already been done: cofactors, such as iron-containing haem, that bind weakly to proteins have been replaced by geometrically similar synthetic catalysts10,11 simply by mixing the artificial cofactor and the apo-enzyme, without scaffold proteins. Whether the intricacies of apo-HydA1 will recognize and accept a simple synthetic [2Fe] subsite analogue alone is not yet known.

The discovery of an active, semi-synthetic variant of HydA1 is an exciting result. It demonstrates that the complex maturase machinery used to construct the enzyme's active site can be circumvented, and provides researchers with a simple system for producing active [FeFe]-hydrogenases from various organisms. It also means that inorganic chemists were on the right track with their models of the [2Fe] subsite, even though those models had low catalytic activity — Berggren and colleagues' findings reveal that although a nitrogen in the bridgehead is crucial for hydrogen formation, the subsite cannot function optimally until it is incorporated into the proper protein cavity.


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    *This article and the paper under discussion1 were published online on 26 June 2013.


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Correspondence to Ryan D. Bethel or Marcetta Y. Darensbourg.

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Bethel, R., Darensbourg, M. Enzymes activated by synthetic components. Nature 499, 40–41 (2013).

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