Abstract

Only a few enzymes are known to utilize stable protein-based radicals as part of their catalytic cycles.1 Among these are both the aerobic and anaerobic ribonucleotide reductases and pyruvate formate-lyase from Escherichia coli. Aerobic and anaerobic ribonucleotide reductases utilize tyrosyl and glycyl radicals, respectively, to generate a transient active site thiyl radical that effects hydrogen atom abstraction in the initial step of ribonucleotide reduction.2 Pyruvate formate-lyase (PFL) has been shown to require a stable glycyl radical to effect the rearrangement of pyruvate to formate.3 The mechanism of generation of these catalytically essential radicals is of considerable interest, but has only been elucidated in any detail for aerobic ribonucleotide reductase. For PFL, an iron-dependent activating enzyme (PFL-AE) is required for activation under reducing conditions in the presence of DTT, via an (S)-adenosylmethionine-dependent hydrogen atom abstraction, to generate a glycyl radical.4,5 However, the nature of the iron center in PFL-AE has until now remained elusive. We report here the first evidence for the presence of an iron-sulfur cluster in PFLAE. When a combination of absorption, variable temperature magnetic circular dichroism (VTMCD), EPR, and resonance Raman (RR) spectroscopies is used, anaerobically prepared PFLAE is shown to contain a mixture of diamagnetic [2Fe-2S]2+ and [4Fe-4S]2+ clusters. Since only [4Fe-4S]2+ clusters remain in dithionite-reduced samples and (S)-adenosylmethionine (SAM) is required to effect reduction to the [4Fe-4S]+ state, these results are interpreted in terms of a subunit-bridging [4Fe-4S]2+,+ cluster in active PFL-AE and suggest that this cluster is involved with generating the 5′-deoxyadenosyl radical from SAM. Purified PFL-AE6 has a distinct red-brown color and a UVvis absorption spectrum consistent with the presence of an Fe-S cluster (Figure 1a). Analysis of four distinct preparations indicated 1.5 ( 0.1 mol of iron and 1.7 ( 0.2 mol of acidlabile sulfide per mole of enzyme monomer.7 The specific activity of purified PFL-AE containing 1.5 mol of iron per mole of enzyme was 3800 U/mg, compared to 540 U/mg for enzyme with a low cluster content (<0.2 mol of iron per mole of enzyme), indicating a direct correlation between cluster content and enzyme activity.8,9 In common with most biological Fe-S centers, reduction with dithionite results in partial bleaching of the visible absorption. However, in contrast to all known types of Fe-S centers which are paramagnetic in at least one oxidation state, neither the as-purified or dithionite-reduced samples contain a paramagnetic Fe-S cluster, as evidenced by parallel and perpendicular X-band EPR and VTMCD studies over the temperature range of 4-50 K.10 The identity of the diamagnetic Fe-S clusters in these samples was revealed by RR studies in the Fe-S stretching region (Figure 1b). Although the signal-to-noise ratio is poor due to high background fluorescence, the RR spectrum of

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