Electron-Nuclear Double Resonance Spectroscopic

Evidence
That S-Adenosylmethionine Binds in Contact with
the
Catalytically Active [4Fe-4S]+ Cluster of Pyruvate
Formate-Lyase Activating Enzyme

Charles J. Walsby, Wei Hong,William E. Broderick, Jennifer Cheek,

Danilo Ortillo, Joan B. Broderick, and Brian M. Hoffman

Presented by: Nathaly Murillo

 Overview

Introduction
●

Materials
●

● Methods

Data
●

Conclusion
●
 Introduction
● Pyruvate formate-lyase
activating enzyme (PFL-AE)
utilizes iron-sulfur clusters and
S-adenosylmethionine (AdoMet)
to initiate radical catalysis
● Stable glycyl radical at G734 of
PFL
● Reductive cleavage of AdoMet
● Yields methionine and 5'-
deoxyadenosine (5'-dAdo)
● PFL converts pyruvate and CoA
to formate and acetyl-CoA
 Introduction
● Purified PFL-AE contains mostly cuboidal
[3Fe-4S]+ which are converted to [4Fe-4S]
2+/+ clusters in reducing conditions.
● The [4Fe-4S]+ form is the essential and
active form
● Carried out EPR and ENDOR studies of
the PFL-AE -AdoMet complex to monitor
the interaction of AdoMet with the [4Fe-
4S] cluster
 Materials and Methods
● PFL-AE was purified from E. coli
● AdoMet was labeled with 2H (CD3-AdoMet)
● 87.9% yield
● And with 13 C (13 CH3-AdoMet)
● 86.0% yield
● Prepared [4Fe-4S]+PFL-AE for EPR and
[4Fe-4S]2+PFL-AE for ENDOR analysis
 EPR Results
● A: PFL-AE [4Fe-
4S]1+ without
AdoMet
● B: PFL-AE [4Fe-
4S]1+with bound
AdoMet
2
H ENDOR Data
(a) and (d)
photoreduced
sample;
(b) cryoreduced
sample
(c) and (e)
Simulations (dashed
lines) with dipolar A
tensors
 13
C ENDOR DATA

(a) with methyl-13C-AdoMet, photoreduced, at g, (b) with

methyl-13C-AdoMet, (d) with methyl-13C-AdoMet,
photoreduced, at g|
Model for the interaction of AdoMet with the [4Fe-4S] cluster of PFL-AE
 Conclusion
● EPR spectroscopy provides evidence that AdoMet might
be closely associated with the Iron-Sulfur cluster of PFL-
AE
● The spectrum of AdoMet is identical in the + and+2 iron-
sulfur clusters for both EPR and ENDOR
● Substantial 2H and 13C hyperfine couplings from labeled
AdoMet bound to the + cluster demonstrates binding
adjacent to the 4Fe cluster.
● the existence of an isotropic contribution to the 13C
tensor requires that there be overlap between orbitals on
the cluster and on AdoMet,
● The sulfonium of the AdoMet interacts with a sulfur not
an iron of the cluster.
● The iron in the cluster may displace ligands present in
the absence of AdoMet