Transcriptional control of the nitrogen fixation (nif) genes in response to oxygen in Azotobacter vinelandii is mediated by nitrogen fixation regulatory protein L (NifL), a regulatory flavoprotein that modulates the activity of the transcriptional activator nitrogen fixation regulatory protein A (NifA). CD spectra of purified NifL indicate that FAD is bound to NifL in an asymmetric environment and the protein is predominantly α-helical. The redox potential of NifL is -226 mV at pH 8 as determined by the enzymic reduction of NifL by xanthine oxidase/xanthine in the presence of appropriate mediators. The reduction of NifL by xanthine oxidase prevented NifL from acting as an inhibitor of NifA. In the absence of electron mediators NifL could also be reduced by Escherichia coli flavohaemoprotein (Hmp) with NADH as reductant. Hmp contains a globin-like domain with haem B as prosthetic group and an FAD-containing oxidoreductase module. The carboxyferrohaem form of Hmp was competent to reduce NifL, suggesting that electron donation to NifL originates from the flavin in Hmp rather than by direct electron transfer from the haem. Spinach ferredoxin:NAD(P) oxidoreductase, which adopts a folding similar to the FAD- and NAD-binding domains of Hmp, also reduced NifL with NADH as reductant. Re-oxidation of NifL occurs rapidly in the presence of air, raising the possibility that NifL might sense intracellular oxygen. We propose a physiological redox cycle in which the oxidation of NifL by oxygen and hence the activation of its inhibitory properties occurs rapidly, in contrast with the switch from the active to the reduced form of NifL, which occurs more slowly.

Avicins are a recently discovered family of plant-derived terpenoid molecules that possess proapoptotic, antiinflammatory, and cytoprotective properties in mammalian cells. Previous work demonstrating that avicins can exert their effects by suppressing or activating the redox-sensitive transcription factors NF-B and nuclear factor-erythroid 2 p45-related factor (Nrf2), respectively, has raised the idea that they may react with critical cysteine residues. To understand the molecular mechanism through which avicins regulate protein function, we examined their effects on the paradigmatic redox-responsive transcriptional activator, OxyR of Escherichia coli, which protects bacterial cells against oxidative and nitrosative stresses. In vitro transcription assays demonstrated that avicins activate OxyR and its target genes katG and oxyS in a DTT-reversible manner. In addition, katG-dependent hydroperoxidase I activity was enhanced in avicin-treated bacteria. Mass spectrometric analysis of activated OxyR revealed thioesterification of the critical regulatory cysteine, Cys-199, to an avicin fragment comprising the outer monoterpene side chain. Our results indicate that avicinylation can induce adaptive responses that protect cells against oxidative or nitrosative stress. More generally, transesterification may represent a previously undescribed thioldirected posttranslational modification, which extends the code for redox regulation of protein function.cysteine ͉ terpenes ͉ thioester ͉ OxyR ͉ redox C lassic signal transduction is characterized by ligand-induced stimulation of pathways comprised of interconnected groups of proteins that undergo various regulatory modifications. Redox-based signaling usually involves small reactive molecules, which covalently and reversibly modify specific cysteine targets to regulate multiple cell processes, including cell proliferation, differentiation, and apoptosis (1). Recent work has demonstrated a significant interface that connects a prototypic redoxrelated signaling process, S-nitrosylation, with ligand-induced signaling involving multiprotein complexes (2, 3), and accumulating evidence suggests that malfunction in redox-based signaling may underlie disease pathogenesis (4-7). Hence, redox regulation may play an important role in normal physiology, as well as in a growing list of medical disorders.Cells adapt to various forms of stress in part by activating redox-responsive genes, which encode proteins involved in detoxification, repair, and maintenance of homeostasis. Several classes of redox-sensitive transcription factors have been shown to regulate the expression of these genes (8). Studies designed to elucidate the mechanism by which such transcription factors are regulated have revealed the importance of critical cysteine residues, which play sensory as well as regulatory roles in transcriptional responses. In particular, cysteine thiols in transcription factors have been shown to exhibit highly selective and differential reactivity toward different reactive oxygen or nitrog...