M C Kennedy

DNA BINDING AND DIMERIZATION OF THE FE-S-CONTAINING FNR PROTEIN FROM ESCHERICHIA COLI ARE REGULATED BY OXYGEN

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 oxygen-stable 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 S2−/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.

Engineering of protein bound iron‐sulfur clusters

An increasing number of iron-sulfur (Fe-S) proteins are found in which the Fe-S cluster is not involved in net electron transfer, as it is in the majority of Fe-S proteins. Most of the former are (de)hydratases, of which the most extensively studied is aconitase. Approaches are described and discussed by which the Fe-S cluster of this enzyme could be brought into states of different structure, ligation, oxidation and isotope composition. The species, so obtained, provided the basis for spectroscopic and chemical investigations. Results from studies by protein chemistry, EPR, Mössbauer, 1H, 2H and 57Fe electron-nuclear double resonance spectroscopy are described. Conclusions, which bear on the electronic structure of the Fe-S cluster, enzyme-substrate interaction and the enzymatic mechanism, were derived from a synopsis of the recent work described here and of previous contributions from several laboratories. These conclusions are discussed and summarized in a final section.

Aconitase: An Iron—Sulfur Enzyme

Publisher Summary This chapter describes an iron–sulfur enzyme—aconitase. Although the presence and function of the Krebs cycle enzyme aconitase has been known for over 50 years, it is only recently that many of the unusual properties of this protein can be understood in molecular terms. Through the application of spectroscopy, especially Mossbauer (MB) spectroscopy in combination with electron paramagnetic resonance (EPR), in addition to chemical methods, it was established that the activation process involved the conversion of a [3Fe–4S] cluster to a [4Fe–4S] form. These earlier studies demonstrated that the iron added during this reaction, labeled Fe a , was site specific. These discoveries in the early 1980s allowed for the subsequent design of a number of experiments to investigate the details of the role of the cluster in the catalytic mechanism of the enzyme. Aconitase and ferredoxin (Fd) II of Desulfovibrio gigas ( D. gigas ) were among the first proteins that were shown to contain a 3Fe cluster. Because of its low molecular mass and its stability, Fd II of D. gigas became the prime example for spectroscopic characterization of proteins containing 3Fe clusters.