The role and properties of the Fe-S clusters in spinach ferredoxin-thioredoxin reductase (FTR), E. coli biotin synthase and E. coli pyruvate formatelyase activating enzyme (PFL-AE) have been investigated by the combination of EPR, resonance Raman, and UV-visible absorption and variable temperature magnetic circular dichroism spectroscopies. FTR is shown to be a novel class of disulfide reductase with an active site involving a [4Fe-4S] cluster and an adjacent cysteine disulfide. The results suggest that the cluster stabilizes the one-electron reduced intermediate of the enzymatic reaction by forming a covalent adduct with one of the cysteines of the active site disulfide, leaving the other cysteine available to form the heterodisulfide adduct with the substrate disulfide. Biotin synthase and PFL-AE are both shown to be homodimeric enzymes with subunit-bridging [4Fe-4S] clusters that undergo novel [4Fe-4S]2+ ⇄ 2[2Fe-2S]2+ + 2e- cluster conversions. It is proposed that the [4Fe-4S] clusters in both enzymes are directly involved with generating a 5′-deoxyadenosyl radical from S-adenosyl-L-methionine and that the oxidative cluster conversion provides a means of regulating enzyme activity on exposure to O2 without irreversible cluster degradation. The possibility that the catalytic roles for the [4Fe-4S] clusters in both classes of enzyme investigated in this work involve novel μ3-S2--based cluster chemistry is discussed.
Bibliographical noteFunding Information:
We are indebted to our collaborators, Peter Schurmann and coworkers, University of Neuchbtel, Switzerland (spinach FTR), Dennis Flint and coworkers, E. I. du Pont de Nemours, Wilmington, USA (biotin synthase), and Joan Broderick and coworkers, Amherst College, Amherst, USA (PFL-AEi), for supplying enzyme samples for spectroscopic investigations and for numerous insightfil discussions. The spectroscopic studies summarized in this article were supported by a grant from NIH (GM-51962 to M.K.J.)