Understanding the Role of [4Fe-4S] Clusters in Hydroxylation during the Anaerobic Biosynthesis of Ubiquinone

Abstract

Ubiquinone is an important biological cofactor which is responsible electron transporter in cellular respiration. The biosynthesis of ubiquinone in Escherichia coli occurs via both aerobic and anaerobic pathways, offering a metabolic flexibility that may aid in rapid adaptation to varying oxygen concentrations. In this biosynthetic pathway, the precursor octaprenylphenol, is hydroxylated three times. The hydroxylations in the anaerobic pathway are performed by two U32 proteins, UbiU and UbiV, which rely on iron-sulfur clusters for catalytic activity. In this work, I characterize the native clusters of both UbiU and UbiV as [4Fe-4S] clusters via CW EPR spectroscopy and UV-visible absorption spectroscopy. In the reduced form, UbiV exists as a mixed population of S=1/2 and S=3/2 ground spin states, whereas UbiU is only in the S=1/2 state. Both clusters are vulnerable to loss of an iron to form a [3Fe-4S] cluster under oxidizing conditions, and both clusters react with the strong ligand cyanide, indicating one iron site in the cluster that is coordinatively unsaturated or has an easily dissociated ligand. Upon incubation with a substrate phenol analogue and the native co-substrate prephenate, a new EPR signal is observed along with a decrease in intensity of the [4Fe-4S]1+ signal, suggesting cluster-substrate interaction or cluster decomposition. Understanding of the electronic structure of these clusters and their reactivity offers insight into the unprecedented mechanisms of anaerobic hydroxylation by U32 hydroxylases.