The ferredoxin-like domain of the activating enzyme is required for generating a lasting glycyl radical in 4-hydroxyphenylacetate decarboxylase.

Abstract

4-Hydroxyphenylacetate decarboxylase-activating enzyme (4Hpad-AE) uses S-adenosylmethionine (SAM or AdoMet) and a [4Fe-4S] ²⁺/⁺cluster (RS cluster) to generate a stable glycyl radical on the decarboxylase. 4Hpad-AE might bind up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert C-terminal to the RS cluster-binding motif. Except for the AEs of pyruvate formate-lyase and anaerobic ribonucleotide reductase, all glycyl radical-activating enzymes possess a similar ferredoxin-like domain, whose functional role is still poorly understood. To assess the role of the putative ferredoxin clusters from 4Hpad-AE, we combined biochemical and spectroscopic methods to characterize a truncated version of the protein (Δ66-AE) devoid of the ferredoxin-like domain. We found that Δ66-AE is stable, harbors a fully active RS cluster and can activate the decarboxylase. From the similar cleavage rates for S-adenosylmethionine of Δ66-AE and wild-type AE, we infer the reactivity of the RS cluster is unperturbed by the absence of the ferredoxin-like domain. Thus, the auxiliary clusters are not required as electron conduit to the RS cluster for effective reductive cleavage of SAM. The activation of the decarboxylase by Δ66-AE is almost as fast as with wild-type AE, but the generated glycyl radical is short living. We postulate that the ferredoxin-like domain is not required for SAM-dependent glycyl radical generation in the decarboxylase, but is necessary for producing a lasting glycyl radical.