Spectroscopic Investigation of Pyruvate Formate Lyase-activating Enzyme: A Look into EPR, ENDOR and Mossabuer Spectroscopy

Danilo O Ortillo,Joan B Broderick

doi:10.19026/rjaset.8.1072

Abstract

Electron Paramagnetic Resonance (EPR) and Electron Nuclear Double Resonance (ENDOR) spectroscopies are extremely powerful and versatile methods for the characterization of paramagnetic systems in biology, chemistry and physics. For iron centers in the radical SAM enzymes however, Mossbauer spectroscopy has proven to be both powerful and useful as a complementary spectroscopic technique in determining not just the oxidation states but also the type of iron species present in the catalytic center. The cluster content of the radical SAM protein, Pyruvate Formate-Lyase-Activating Enzyme (PFL-AE), was characterized using EPR and Mossbauer techniques while additional ENDOR analysis helped determine the novel interaction of the co-substrate, SAdenosylmethionine (SAM or AdoMet) with the Fe-S cluster of PFL-AE. The anchoring role of the Fe-S cluster to the co-substrate derived from the spectroscopic data supports the mechanism where a SAM-based radical species is involved during catalysis.

Highlights

The chemistry of Fe-S clusters has been thoroughly documented through the years and most of the basic features that we observe with the protein-bound clusters have been determined via in vitro studies using model clusters, generally not in aqueous medium and mostly in anaerobic conditions (Berg and Holm, 1982; Holm, 1992)
Cheek and Broderick (2001) have shown that Pyruvate Formate-Lyase-Activating Enzyme (PFL-AE) purified under anaerobic reducing conditions in the presence of DTT yield essentially Electron Paramagnetic Resonance (EPR)-silent clusters, presumably in the (4Fe-4S)2+ state, which can be readily reduced to (4Fe-4S)+, the cluster that is responsible for providing the electron necessary for AdoMet dependent glycyl radical generation on Pyruvate Formate-Lyase (PFL)
Analysis of the Mössbauer data of the as-isolated PFL-AE shows the presence of mixtures of Fe-S clusters

Summary

INTRODUCTION

The chemistry of Fe-S clusters has been thoroughly documented through the years and most of the basic features that we observe with the protein-bound clusters have been determined via in vitro studies using model clusters, generally not in aqueous medium and mostly in anaerobic conditions (Berg and Holm, 1982; Holm, 1992). Cheek and Broderick (2001) have shown that PFL-AE purified under anaerobic reducing conditions in the presence of DTT yield essentially EPR-silent clusters, presumably in the (4Fe-4S)2+ state, which can be readily reduced to (4Fe-4S)+, the cluster that is responsible for providing the electron necessary for AdoMet dependent glycyl radical generation on PFL. Henshaw et al (2000) have demonstrated that the (4Fe-4S)+ is the catalytically active cluster of PFL-AE and that it donates the electron required for reductive cleavage of AdoMet. The reduced cluster in the presence of AdoMet shows a nearly axial signal shown in Fig. 3B which is a result of one of the unique axis being different from the other two = ˧ ≠ ˧ {. Hyperfine structure of Fe-mossbauer spectra: The Hamiltonian of the nucleus can be described by a sum of the unperturbed nuclear Hamiltonian and a perturbation caused by the hyperfine interactions which are present at the nucleus of the Mossbauer isotope:

Because Mlattice is much larger than the nuclear

Findings

CONCLUSION