Radical SAM' enzymes generate catalytic radicals by combining a 4Fe±4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the ®rst crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 A Ê resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe±4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A Ê ) and a neighboring sulfur of the cluster (3.6 A Ê ), allowing single electron transfer from the 4Fe±4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5¢-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the ®rst. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V-shaped glutamyl-tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the ''Radical SAM enzyme'' coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure-function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.

Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.Fe-S clusters perform important functions in multiple cellular processes, including respiration, gene regulation, and the trichloroacetic acid cycle (1). In vivo Fe-S biogenesis requires specific proteins such as a pyridoxal phosphate-dependent cysteine desulfurase that mobilizes sulfur from L-cysteine and an Fe-S cluster scaffold upon which the nascent cluster can be constructed prior to its transfer to an apoprotein. Homologues of these two core components can be found in numerous organisms ranging from bacteria to humans (1-3). In bacteria the basal pathway of Fe-S cluster assembly is known as Isc 5 (ironsulfur cluster assembly) (4, 5).Fe-S cluster homeostasis is sensitive to disruption by reactive oxygen and reactive nitrogen species or by iron limitation (6 -8). Recently it was discovered that biosynthesis of Fe-S clusters during adverse stress conditions such as iron starvation and oxidative stress requires a specialized assembly pathway different from the basal Isc system. This pathway, termed the Suf pathway (mobilization of sulfur), is regulated in response to those stress conditions (7-9). The Suf pathway is present in cyanobacteria and the chloroplast of higher plants as well as in bacteria, including human pathogens such as Yersinia pestis and Mycobacterium tuberculosis. In M. tuberculosis, the Suf pathway seems to be the primary Fe-S cluster assembly system, as deleting the suf genes is thought to be lethal to that organism (10).Because of its importance, Suf has been the focus of intense study at the biochemical level, especially in the Gram-negative bacterium Escherichia coli. The sufABCDSE operon in E. coli encodes six proteins. SufA is homologous to IscA in the basal Isc Fe-S cluster pathway. The exact function of SufA is unknown. Although it has been suggested to act as an Fe-S scaffold, it cannot be excluded that SufA pla...

In bacteria the oxygen-independent coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of coproporphyrinogen-III to protoporphyrinogen-IX. The Escherichia coli hemN gene encoding a putative part of this enzyme was overexpressed in E. coli. Anaerobically purified HemN is a monomeric protein with a native M r ‫؍‬ 52,000 ؎ 5,000. A newly established anaerobic enzyme assay was used to demonstrate for the first time in vitro coproporphyrinogen-III oxidase activity for recombinant purified HemN. The enzyme requires S-adenosyl-L-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis. An oxygen-sensitive iron-sulfur cluster was identified by absorption spectroscopy and iron analysis.

The biogenesis of iron-sulfur [Fe-S] clusters requires the coordinated delivery of both iron and sulfide. Sulfide is provided by cysteine desulfurases that use L-cysteine as sulfur source. So far, the physiological iron donor has not been clearly identified. CyaY, the bacterial ortholog of frataxin, an iron binding protein thought to be involved in iron-sulfur cluster formation in eukaryotes, is a good candidate because it was shown to bind iron. Iron-sulfur [Fe-S] clusters are ubiquitous and evolutionary ancient prosthetic groups that are required to sustain fundamental life processes. They are involved in electron transfer, substrate binding/activation, iron/sulfur storage, regulation of gene expression, and enzyme activity reactions (1). Formation of intracellular [Fe-S] clusters does not occur spontaneously but requires a complex biosynthetic machinery. In Escherichia coli three different types of [Fe-S] cluster biosynthesis systems have been identified so far, namely the iron-sulfur cluster, sulfur mobilization, and cysteine sulfinate desulfinase systems (2, 3). These different machineries have in common the involvement of a cysteine desulfurase that allows utilization of cysteine as source of sulfur atoms Important questions related to [Fe-S] cluster biosynthesis include: (i) the molecular mechanism by which iron and sulfide are assembled on the scaffold protein; (ii) how accessory proteins (chaperones in particular) participate in the process; and (iii) how the cluster is transferred from the scaffold to an apo target protein. Another essential question is the identity of iron and sulfur donors for the formation of [Fe-S] clusters. Whereas L-cysteine has been identified as the ultimate source of sulfur, the question "Where does the iron come from?" still remains unanswered. Whereas some preliminary answers have been provided to most of the above issues, very little is known regarding the last question. It is simply assumed that, because of its toxicity, iron has to be stored and transported by proteins from which it can be mobilized for assembly of iron sites. In bacteria, IscA and YggX, which were shown to be able to bind iron and to be, to some extent, involved in [Fe-S] metabolism, are potential candidates requiring further investigations (9 -12). However, this is controversial because IscA was proposed to be an [Fe-S] scaffold protein (5, 7), whereas a recent report could not establish iron binding to YggX (13).More information concerning a putative iron donor protein is available in eukaryotic systems. In eukaryotes, [Fe-S] cluster assembly requires two biosynthetic protein machineries. One is localized in the mitochondria and functions in the assembly of all cellular [Fe-S] proteins, whereas the other one is cytosolic, specifically involved in the maturation of cytosolic and nuclear [Fe-S] (15); (ii) the yeast frataxin (Yfh1p) is involved in the regulation of iron homeostasis (16,17) and is required for maturation of [Fe-S] cluster containing proteins (18), and its inactivation results in i...

Assembly of iron-sulfur (Fe-S) clusters and maturation ofFe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC 2 D complex, only in its reduced form (FADH 2 ), which has the ability to reduce ferric iron. These results suggest that the SufBC 2 D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH 2 -dependent reductive mobilization of iron.

SummaryMethane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilisation of methane through anaerobic methane oxidation. The enzyme employs an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a novel methyl radical/Ni(II)-thiolate intermediate. However, the biosynthesis of coenzyme F430 from the common primogenitor uroporphyrinoge III, incorporating 11 steric centres into the macrocycle, has remained poorly understood although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamisation and carbocyclic ring formation. We have now identified the proteins that catalyse coenzyme F430 biosynthesis from sirohydrochlorin, termed CfbA-E, and shown their activity. The research completes our understanding of how nature is able to construct its repertoire of tetrapyrrole-based life pigments, permitting the development of recombinant systems to utilise these metalloprosthetic groups more widely.

SummaryThe oxygen regulator Fnr is part of the regulatory cascade in Bacillus subtilis for the adaptation to anaerobic growth conditions. In vivo complementation experiments revealed the essential role of only three cysteine residues (C227, C230, C235) at the Cterminus of B. subtilis Fnr for the transcriptional activation of the nitrate reductase operon ( narGHJI ) and nitrite extrusion protein gene ( narK ) promoters. UV/ VIS, electron paramagnetic spin resonance (EPR) and Mössbauer spectroscopy experiments in combination with iron and sulphide content determinations using anaerobically purified recombinant B. subtilis Fnr identified the role of these three cysteine residues in the formation of one