46065-AC4
The Functionalization of Unactivated Alkanes by the Radical SAM Superfamily of Enzymes
The radical SAM (RS) superfamily of enzymes is a newly described class of metalloproteins that employ a unique [4Fe–4S]2+/+–S-adenosyl-L-methionine (SAM) cofactor to initiate and carry out catalysis by mechanisms involving obligate radical intermediates. Three of the iron atoms of the [4Fe–4S] cluster are coordinated by one of three conserved cysteinyl residues residing in a CxxxCxxC motif, common to almost all enzymes within this superfamily. The fourth iron, which is not ligated by a protein-derived cysteinyl residue, contains open coordination sites to which SAM binds via its α-amino and α-carboxylate groups. The function of the iron–sulfur (Fe/S) cluster is to inject an electron into the sulfonium group of SAM, causing it to fragment into L-methionine and a 5'-deoxyadenosyl 5'-radical, an obligate intermediate in RS enzymes. This intermediate then initiates catalysis by abstracting a hydrogen atom from the appropriate substrate. Relatively little attention has been given to understanding the energetics of forming the high-energy 5'-dA• that is responsible for initiating catalysis. Redox potential determinations have only been carried out on a couple of RS enzymes, and the midpoint potentials of the bound Fe/S clusters are typically ~-550 mV, which is significantly higher (i.e., less favorable) than that required to inject an electron into the sulfonium of SAM (~-1.3 V), suggesting that compensatory interactions involving the enzyme and the relevant substrates and cofactors must be operative. The goal of the grant from the ACS-PRF was to try to determine what these compensatory interactions are for several RS enzymes, notably lipoyl synthase (LS) and biotin synthase (BS). Several specific aims were noted: 1. To conduct X-ray absorption spectroscopy (XAS) studies on LS and BS in collaboration with Dr. Robert Scott (University of Georgia) to assess whether the sulfur of methionine becomes coordinated to one of the irons of the Fe/S cluster concomitant with reductive cleavage of SAM, and to determine whether coordination correlates with the ability of the substrate to induce cleavage of SAM. 2. To assess whether the binding of SAM and SAM analogs to the [4Fe–4S] cluster in LS and BS alters the midpoint potential of the cluster. 3. To use ENDOR spectroscopy in collaboration with Brian Hoffmann to probe the relative orientation of the methionine group of SAM in the active site of LS using analogs of SAM that are unable to form the N/O chelate. During the initial year of funding we made significant progress with specific aims 1 and 2, and are eager to begin studies on specific aim 3. As stated, the goal of specific aim 1 is to assess whether the sulfur atom of methionine becomes coordinated to one of the irons of the Fe/S cluster concomitant with reductive cleavage of SAM. In the enzyme lysine 2,3-aminomutase, the PI was involved in experiments to study the mechanism of cleavage of SAM. This enzyme catalyzes a reversible cleavage of SAM to generate the 5'-dA•; however, at the end of each turnover the 5'-dA• recombines with L-met to reform SAM with concomitant reduction of the Fe/S cluster by one electron. Given the unfavorable energetics associated with transferring an electron from a typical [4Fe–4S] cluster to SAM, it was hypothesized that concomitant coordination of the cleavage product, L-met, to the cluster might compensate somewhat for the unfavorable equilibrium. Indeed, XAS was used in combination with the selenium-containing analog of SAM, Se-adenosyl-L-selenomethionine, to show that the product of cleavage, L-selenomethionine, coordinates to an iron on the Fe/S cluster. This was manifested by a 2.7 Å interaction after fitting the XAS data appropriately. Importantly, this same interaction could be recapitulated by incubating the enzyme with L-selenomethionine and 5'-dA, but only in the presence of substrate. The substrate dependence of the interaction is suggestive of its relevance in catalysis. Our initial goals outlined in this grant were to repeat the above XAS experiments on LS and BS to determine whether this coordination subsequent to cleavage is a general phenomenon, and is therefore required for cleavage to transpire. We indeed performed these experiments in collaboration with Dr. Robert Scott's lab. In contrast to lysine 2,3-aminomutase, observation of the 2.7 Å interaction in the presence of L-selenomethionine and 5'-dA took place in the absence of substrate. In fact, addition of substrate caused the interaction to disappear. The second specific aim was to assess whether the binding of SAM to the [4Fe–4S] cluster in LS and BS alters its redox potential. A big part of this specific aim was to build a spectroelectrochemical titrator to be used to determine the midpoint potentials of the relevant [4Fe–4S] clusters. We have indeed done this according to the specifications in a publication by Hinckley and Frey. We are now in the process of carrying out these experiments.
