Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

Abstract

Pyruvate ferredoxin oxidoreductase (PFOR) 1 is a thiamine pyrophosphate (TPP)-dependent enzyme that catalyzes the anaerobic oxidation of pyruvate (with CoA) to acetyl-CoA, CO 2 , and two electrons that are transferred to ferredoxin (eq 1). 2 In this paper, the PFOR electrontransfer mechanism is examined. Early steps in the catalytic cycle follow the Breslow 3 mechanism of TPP activation. After deprotonation, the carbanion at C-2 of TPP reacts with pyruvate to form 2-lactyl-TPP, which decarboxylates to generate CO 2 and the resonant enamine/carbanion intermediate, hydroxyethylidene-TPP (HE-TPP). The fate of this intermediate differs among the TPP-dependent enzymes. In PFOR, oxidation of the intermediate is linked to reduction of an external electron acceptor, for example, the clostridial ferredoxin, which contains two [4Fe-4S] clusters.(1)After formation of HE-TPP, the next step in the PFOR mechanism is one-electron transfer from HE-TPP to one of three [4Fe-4S] 2+ clusters, which generates a paramagnetic [4Fe-4S] 1+ cluster and an HE-TPP radical. 4 This paramagnetic intermediate forms at a rate constant of 140 s −1 at 10 °C (~3000 s −1 at the growth temperature of 55 °C). 4 On the basis of the PFOR structure, 5 cluster A, located only 9.5 Å from the thiazole sulfur of HE-TPP, is one of three [4Fe-4S] clusters that are separated by 10-12 Å (Figure 1). 6 Since biological electron transfer is facile whenever the donor and acceptor are within ~15 Å, clusters A (proximal), B (medial), and C (distal, ~4 Å from the surface) are ideally positioned to sequentially transfer electrons to the surface and the external ferredoxin. Cluster A is logically the first to be reduced, since clusters B and C are >20 Å from the radical center on HE-TPP. In the pulsed ELDOR experiment, the primary electron spin-echo (ESE) signal of the [4Fe-4S] 1+ cluster was observed, while the pumping microwave pulse was in resonance with the HE-TPP radical. The Fourier transform (FT) spectrum of the time-domain ELDOR trace exhibits a prominent peak at 1.5 MHz and a smaller feature at 2.7 MHz (trace 1 in Figure 2). Although this spectral shape is reasonably close to that expected in a situation of complete orientational disorder, some orientational selectivity caused by the g-anisotropy of the [4Fe-4S] 1+ center cannot be excluded. To remove the ambiguity, a RIDME experiment was performed. 11 In this experiment, we observed a refocused stimulated ESE signal of the HE-TPP radical, while the longitudinal relaxation of the [4Fe-4S] 1+ center (instead of pumping at a different microwave frequency) served the purpose of modifying the local magnetic field for the radical spin. Since the [4Fe-4S] 1+ center at any orientation is subject to longitudinal relaxation, 12 there is no orientational selectivity in the RIDME experiment.The RIDME experiment was performed at 13 and 4.2 K. At 13 K, the cluster relaxes much more rapidly than at 4.2 K. The difference between the two stimulated ESE spectra gave the RIDME spectrum shown by trace 2 in Figure 2. The only reli...

Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth on Oxalate

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A pulsed EPR method to determine distances between paramagnetic centers with strong spectral anisotropy and radicals: The dead-time free RIDME sequence

“…For transition-metal ions, the spectral width is usually larger. Examples for transition-metal DEER refer to those transition-metal ions that have moderate spectral anisotropies, such as Cu(II), where g-values between 2.37 and 2.08 give rise to spectral widths of 70 mT at X-band EPR, [9][10][11][12] or iron-sulfur centers [13]. Most other transition-metal ions have larger g-anisotropies and faster relaxation times even at cryogenic temperatures.…”

On Cu(II)–Cu(II) distance measurements using pulsed electron electron double resonance

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