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Electron Paramagnetic Resonance Spectroscopic Identification of the Fe–S Clusters in the SPASM Domain-Containing Radical SAM Enzyme PqqE
journal contribution
posted on 2019-12-11, 16:42 authored by Lizhi Tao, Wen Zhu, Judith P. Klinman, R. David BrittPyrroloquinoline quinone (PQQ) is an important redox
active quinocofactor
produced by a wide variety of bacteria. A key step in PQQ biosynthesis
is a carbon–carbon cross-link reaction between glutamate and
tyrosine side chains within the ribosomally synthesized peptide substrate
PqqA. This reaction is catalyzed by the radical SAM enzyme PqqE. Previous
X-ray crystallographic and spectroscopic studies suggested that PqqE,
like the other members of the SPASM domain family, contains two auxiliary
Fe–S clusters (AuxI and AuxII) in addition to the radical SAM
[4Fe–4S] cluster. However, a clear assignment of the electron
paramagnetic resonance (EPR) signal of each Fe–S cluster was
hindered by the isolation of a His6-tagged PqqE variant
with an altered AuxI cluster. In this work, we are able to isolate
soluble PqqE variants by using a less disruptive strep-tactin chromatographic approach. We have unambiguously identified
the EPR signatures for four forms of Fe–S clusters present
in PqqE through the use of multifrequency EPR spectroscopy: the RS
[4Fe–4S] cluster, the AuxII [4Fe–4S] cluster, and two
different clusters ([4Fe–4S] and [2Fe–2S]) bound in
the AuxI site. The RS [4Fe–4S] cluster, the AuxII [4Fe–4S]
cluster, and the [2Fe–2S] cluster form in the AuxI site can
all be reduced by sodium dithionite, with g tensors of
their reduced form determined as [2.040, 1.927, 1.897], [2.059, 1.940,
1.903], and [2.004, 1.958, 1.904], respectively. The AuxI [4Fe–4S]
cluster that is determined on the basis of its relaxation profile
can be reduced only by using low-potential reductants such as Ti(III)
citrate or Eu(II)-DTPA to give rise to a g1 = 2.104 signal. Identification of the EPR signature for each cluster
paves the way for further investigations of SPASM domain radical SAM
enzymes.