Interaction of the Streptomyces Wbl protein WhiD with the principal sigma factor σHrdB depends on the WhiD [4Fe-4S] cluster

The bacterial protein WhiD belongs to the Wbl family of iron–sulfur [Fe-S] proteins present only in the actinomycetes. In Streptomyces coelicolor, it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from in vitro and in vivo experiments with WhiD from Streptomyces venezuelae (SvWhiD), which differs from S. coelicolor WhiD (ScWhiD) only at the C terminus. We observed that, like ScWhiD and other Wbl proteins, SvWhiD binds a [4Fe-4S] cluster that is moderately sensitive to O2 and highly sensitive to nitric oxide (NO). However, although all previous studies have reported that Wbl proteins are monomers, we found that SvWhiD exists in a monomer–dimer equilibrium associated with its unusual C-terminal extension. Several Wbl proteins of Mycobacterium tuberculosis are known to interact with its principal sigma factor SigA. Using bacterial two-hybrid, gel filtration, and MS analyses, we demonstrate that SvWhiD interacts with domain 4 of the principal sigma factor of Streptomyces, σHrdB (σHrdB4). Using MS, we determined the dissociation constant (Kd) for the SvWhiD–σHrdB4 complex as ~0.7 μM, consistent with a relatively tight binding interaction. We found that complex formation was cluster dependent and that a reaction with NO, which was complete at 8–10 NO molecules per cluster, resulted in dissociation into the separate proteins. The SvWhiD [4Fe-4S] cluster was significantly less sensitive to reaction with O2 and NO when SvWhiD was bound to σHrdB4, consistent with protection of the cluster in the complex

Redox-Sensing Iron–Sulfur Cluster Regulators

Significance: Iron–sulfur cluster proteins carry out multiple functions, including as regulators of gene transcription/translation in response to environmental stimuli. In all known cases, the cluster acts as the sensory module, where the inherent reactivity/fragility of iron–sulfur clusters with small/redox-active molecules is exploited to effect conformational changes that modulate binding to DNA regulatory sequences. This promotes an often substantial reprogramming of the cellular proteome that enables the organism or cell to adapt to, or counteract, its changing circumstances. Recent Advances: Significant progress has been made recently in the structural and mechanistic characterization of iron–sulfur cluster regulators and, in particular, the O2 and NO sensor FNR, the NO sensor NsrR, and WhiB-like proteins of Actinobacteria. These are the main focus of this review. Critical Issues: Striking examples of how the local environment controls the cluster sensitivity and reactivity are now emerging, but the basis for this is not yet fully understood for any regulatory family. Future Directions: Characterization of iron–sulfur cluster regulators has long been hampered by a lack of high-resolution structural data. Although this still presents a major future challenge, recent advances now provide a firm foundation for detailed understanding of how a signal is transduced to effect gene regulation. This requires the identification of often unstable intermediate species, which are difficult to detect and may be hard to distinguish using traditional techniques. Novel approaches will be required to solve these problems

Mass spectrometric studies of Cu(I)-binding to the N-terminal domains of B. subtilis CopA and influence of bacillithiol

CopA is a Cu(I)-exporting transmembrane P1B-type ATPase from Bacillus subtilis. It contains two N-terminal cytoplasmic domains, CopAab, which bind Cu(I) with high affinity and to form higher-order complexes with multiple Cu(I) ions. To determine the precise nature of these species, electrospray ionisation mass spectrometry (ESI-MS) under non-denaturing conditions was employed. Up to 1 Cu per CopAab resulted in Cu coordination to one or both CopAab domains. At >1 Cu/CopAab, two distinct dimeric charge state envelopes were observed, corresponding to distinct conformations, each with Cu6(CopAab)2 as its major form. The influence of the physiologically relevant low molecular weight thiol bacillithiol (BSH) on Cu(I)-binding to CopAab was assessed. Dimeric CopAab persisted in the presence of BSH, with previously undetected Cu7(CopAab)2 and Cu6(CopAab)2(BSH) forms apparent

Mass spectrometric detection of iron nitrosyls, sulfide oxidation and mycothiolation during nitrosylation of the NO sensor [4Fe-4S] NsrR

Identification of RRE-type iron-nitrosyl species formed upon nitrosylation of [4Fe–4S] NsrR.</p

Mass spectrometric identification of intermediates in the O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion in FNR

The iron-sulfur cluster containing protein FNR is the master regulator for the switch between anaerobic and aerobic respiration in Escherichia coli and many other bacteria. The [4Fe-4S] cluster functions as the sensory module, undergoing reaction with O2 that leads to conversion to a [2Fe-2S] form with loss of high affinity DNA-binding. Here we report studies of the FNR cluster conversion reaction using time-resolved electrospray ionization mass spectrometry. The data provide new insight into the reaction, permitting the detection of cluster conversion intermediates and products, including a novel [3Fe-3S] cluster and persulfide coordinated [2Fe-2S] clusters ([2Fe-2S](S)n, where n = 1 or 2). Analysis of kinetic data revealed a branched mechanism in which cluster sulfide oxidation occurs in parallel with cluster conversion, and not as a subsequent, secondary reaction, to generate ([2Fe-2S](S)n species. This methodology shows great potential for broad application to studies of protein cofactorsmall molecule interactions

Diversity of Fe2+ entry and oxidation in ferritins

The essential metal iron presents two major problems for life: it is potentially highly toxic due to its redox activity, and its extremely low solubility in aqueous solution in the presence of O2 can make it hard to acquire and store safely. Ferritins are part of nature’s answer to these problems, as they store iron in a safe but accessible form in all types of cells. How they achieve this has been the subject of intense research for several decades. Here, we highlight recent progress in elucidating the routes by which Fe2+ ions access the catalytic ferroxidase centers, and the mechanisms by which Fe2+ is oxidized. Emerging from this is a picture of diversity, both in terms of Fe2+ entry pathways and the roles played by the structurally distinct diiron ferroxidase centers

Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn

Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+ via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres

Sensing mechanisms of iron–sulfur cluster regulatory proteins elucidated using native mass spectrometry

The ability to sense and respond to various key environmental cues is important for the survival and adaptability of many bacteria, including pathogens. The particular sensitivity of iron–sulfur (Fe–S) clusters is exploited in nature, such that multiple sensor-regulator proteins, which coordinate the detection of analytes with a (in many cases) global transcriptional response, are Fe–S cluster proteins. The fragility and sensitivity of these Fe–S clusters make studying such proteins difficult, and gaining insight of what they sense, and how they sense it and transduce the signal to affect transcription, is a major challenge. While mass spectrometry is very widely used in biological research, it is normally employed under denaturing conditions where non-covalently attached cofactors are lost. However, mass spectrometry under conditions where the protein retains its native structure and, thus, cofactors, is now itself a flourishing field, and the application of such ‘native’ mass spectrometry to study metalloproteins is now relatively widespread. Here we describe recent advances in using native MS to study Fe–S cluster proteins. Through its ability to accurately measure mass changes that reflect chemistry occurring at the cluster, this approach has yielded a remarkable richness of information that is not accessible by other, more traditional techniques