The transcription factor FNR from Escherichia coli regulates transcription of genes in response to oxygen deprivation. To determine how the activity of FNR is regulated by oxygen, a form of FNR had to be isolated that had properties similar to those observed in vivo. This was accomplished by purification of an FNR fraction which exhibited enhanced DNA binding in the absence of oxygen. Iron and sulfide analyses of this FNR fraction indicated the presence of an Fe-S cluster. To determine the type of Fe-S cluster present, an oxygenstable mutant protein LH28-DA154 was also analyzed since FNR LH28-DA154 purified anoxically contained almost 3-fold more iron and sulfide than the wild-type protein. Based on the sulfide analysis, the stoichiometry (3.3 mol of S 2؊ /FNR monomer) was consistent with either one [4Fe-4S] or two [2Fe-2S] clusters per mutant FNR monomer. However, since FNR has only four Cys residues as potential cluster ligands and an EPR signal typical of a 3Fe-4S cluster was detected on oxidation, we conclude that there is one [4Fe-4S] cluster present per monomer of FNR LH28-DA154. We assume that the wild type also contains one [4Fe-4S] cluster per monomer and that the lower amounts of iron and sulfide observed per monomer were due to partial occupancy. Consistent with this, the Fe-S cluster in the wild-type protein was found to be extremely oxygen-labile. In addition, molecular-sieve chromatographic analysis showed that the majority of the anoxically purified protein was a dimer as compared to aerobically purified FNR which is a monomer. The loss of the Fe-S cluster by exposure to oxygen was associated with a conversion to the monomeric form and decreased DNA binding. Taken together, these observations suggest that oxygen regulates the activity of wild-type FNR through the lability of the Fe-S cluster to oxygen.

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. The ability to adapt to changes in oxygen concentrations in the environment is common to many organisms. In the facultative anaerobe, Escherichia coli, the transcription factor FNR ( fumarate nitrate reduction) regulates a network of genes that facilitates adaptation to oxygen deprivation by providing alternative pathways for energy generation (1). Recent data suggest that FNR contains a [4Fe-4S] cluster (2-4) and that this cluster apparently mediates the sensitivity of this transcription factor to oxygen, thus limiting FNR activity to anaerobic conditions. The stoichiometry of iron and labile sulfide relative to the number of cysteine ligands of anaerobically purified FNR is most compatible with the presence of a [4Fe-4S] cluster (3). Iron and sulfide analyses and CD spectra of FNR preparations derived from reconstitution of a cluster into apoprotein also supports this cluster assignment (4). The presence of the Fe-S cluster in the anaerobically purified form of FNR is correlated with an increase in dimerization and specific DNA binding (3), compared with the aerobically purified form that lacks an Fe-S cluster (2, 5, 6). Furthermore, the Fe-S cluster is disrupted by oxygen, and this is correlated with the conversion of FNR into an inactive monomeric protein (3). The loss of this cluster as well as the loss of specific DNA binding upon exposure of FNR to oxygen suggested that the [4Fe-4S] cluster, through its intrinsic instability, serves as an oxygen sensor.This paper reports observations on the path of disassembly of the [4Fe-4S] cluster of FNR in vitro. Little is known about the cluster disassembly mechanism of proteins in the absence of chelators, detergents, chemical oxidants, and other nonphysiological agents. The rapid destruction of the Fe-S cluster of FNR, simply upon exposure of a purified protein solution to air, offers an opportunity to obtain information on the progress and mode of the disassembly of the [4Fe-4S] cluster of this protein. We have used chemical analysis for iron and sulfide as well as electronic, EPR, and Mössbauer spectroscopies to monitor the disassembly process. We report here the unexpected observation that the [4Fe-4S] cluster of FNR is converted in less than 5 min to a [2Fe-2S] cluster in about 60% yield as judged from Mössbauer spectra and this conversion results in a reduction in DNA-binding ability. The [4Fe-4S] 2ϩ cluster can be regenerated from the [2Fe-2S] cluster or its components by reduction with dithionite.

In the facultative anaerobe Escherichia coli, the transcription factor FNR (fumarate nitrate reduction) regulates gene expression in response to oxygen deprivation. To investigate how the activity of FNR is regulated by oxygen availability, two mutant proteins, DA154 and LH28-DA154, which have enhanced in vivo activity in the presence of oxygen, were purified and compared. Unlike other previously exam-ined FNR preparations, the absorption spectrum of LH28-DA154 had two maxima at 324 nm and 419 nm, typical of iron-sulfur (Fe-S)-containing proteins. Consistent with these data, metal analysis showed that only the LH28-DA154 protein contained a significant amount of iron and acid-labile sulfide, and, by low temperature EPR spectroscopy, a signal typical of a [3Fe-4S] + cluster was detected. The protein that contained the Fe-S cluster also contained a higher proportion of dimers and had a 3-to 4-fold higher apparent affinity for the target DNA than the DA154 protein.In agreement with this, we found that when the LH28-DA154 protein was treated with an iron chelator (c,a'-dipyridyl), it lost its characteristic absorption and the apparent affinity for DNA was reduced 6-fold. However, increased DNA binding and the characteristic absorption spectrum could be restored by in vitro reconstitution of the Fe-S center. DNA binding of the LH28-DA154 protein was also affected by the redox state of the Fe-S center, since protein exposed to oxygen bound 1/10th as much DNA as the protein reduced anaerobically with dithionite. The observation that DNA binding is enhanced when the Fe-S center is reduced indicates that the redox state of the Fe-S center affects the DNA-binding activity of this protein and suggests a possible mechanism for regulation of the wild-type protein.A fundamental problem in biology is to understand the biochemical and molecular events that allow cells to sense and adapt to changes in oxygen in the environment. To address this problem, we have been studying how the activity of the Escherichia coli transcription factor FNR (fumarate nitrate reduction) is regulated by oxygen availability. In the absence of oxygen, FNR is converted to an active form that regulates the synthesis of many enzymes. A central question is to determine how FNR is converted to an active form.One region of FNR proposed to be required for regulation by oxygen is located near the N terminus and is characterized by a cluster of four closely spaced cysteine residues (16Cys-Xaa3-Cys-Xaa2-Cys-Xaa5-Cys29). Consistent with its supposed function as an oxygen regulatory site, this N-terminal region is located outside the regions of FNR that are similar to the DNA-binding and cAMP-binding domains of the well-studied, cAMP-dependent CAP protein (1). The FNR cysteine residues appear to be important components of the N-terminal region since mutational analysis has shown that three of the four cysteine residues (Cys-20, -23, -29) as well as one outside this region (Cys-122) are required for function (2).Several studies (3-8) suggest that iron is ...

2ϩ cluster in the presence of O 2 remains to be elucidated (10). Nevertheless, the decrease in site-specific DNA binding and in the extent of dimerization that occurs concomitant with cluster conversion provides a reasonable explanation for why FNR is only active as a transcription factor under anaerobic conditions in vitro (11,13,14). Furthermore, 2ϩ to 2ϩ cluster conversion in FNR has also been observed in vivo following exposure of anaerobically maintained cells to air (15). Thus, inactivation of FNR by O 2 appears to be mediated by an O 2 -sensitive [4Fe-4S] 2ϩ cluster both in vitro and in vivo. Although these investigations have provided clear insights into how FNR from anaerobic cells is inactivated by O 2 , much less is known about how FNR is maintained in an inactive state under aerobic growth conditions. Although WT-FNR purified from aerobically grown cells has previously been shown to lack any Fe-S cluster 17)), it is not known if this apo-FNR arises from the continuous destruction of [4Fe-4S] cluster assembly under these growth conditions. Techniques to distinguish between these two possibilities for WT-FNR are not * This work was supported in part by National Institute of Health Grants GM-45844 (to P. J. K.) and GM-22701 (to E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.† Deceased.