1. Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, D35032 Marburg, Germany
2. Present address: Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK.

Correspondence to: Antonio J. Pierik¹ Correspondence and requests for materials should be addressed to A.J.P. (Email: pierik@staff.uni-marburg.de).

The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO₂, 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions¹. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed² to mediate this reaction catalysed by an iron–sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron–sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases³ and the related benzoyl-CoA reductases⁴—present in anaerobically living bacteria—employ ketyl radical intermediates. The absence of radical generators such as coenzyme B₁₂, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.