Spectroscopic and Computational Investigation of the Epoxyqueuosine Reductase QueG Reveals Intriguing Similarities with the Reductive Dehalogenase PceA

Queuosine
(Q) is a highly modified nucleoside of transfer RNA that
is formed from guanosine triphosphate over the course of eight steps.
The final step in this process, involving the conversion of epoxyqueuosine
(oQ) to Q, is catalyzed by the enzyme QueG. A recent X-ray crystallographic
study revealed that QueG possesses the same cofactors as reductive
dehalogenases, including a base-off Co­(II)­cobalamin (Co­(II)­Cbl) species
and two [4Fe-4S] clusters. While the initial step in the catalytic
cycle of QueG likely involves the formation of a reduced Co­(I)­Cbl
species, the mechanisms employed by this enzyme to accomplish the
thermodynamically challenging reduction of base-off Co­(II)­Cbl to Co­(I)­Cbl
and to convert oQ to Q remain unknown. In this study, we have used
electron paramagnetic resonance (EPR) and magnetic circular dichroism
(MCD) spectroscopies in conjunction with whole-protein quantum mechanics/molecular
mechanics (QM/MM) computations to further characterize wild-type QueG
and select variants. Our data indicate that the Co­(II)­Cbl cofactor
remains five-coordinate upon substrate binding to QueG. Notably, during
a QM/MM optimization of a putative QueG reaction intermediate featuring
an alkyl–Co­(III) species, the distance between the Co ion and
coordinating C atom of oQ increased to >3.3 Å and the C–O
bond of the epoxide reformed to regenerate the oQ-bound Co­(I)­Cbl reactant
state of QueG. Thus, our computations indicate that the QueG mechanism
likely involves single-electron transfer from the transient Co­(I)­Cbl
species to oQ rather than direct Co–C bond formation, similar
to the mechanism that has recently been proposed for the tetrachloroethylene
reductive dehalogenase PceA.