Properties of the iron-sulfur cluster electron transfer relay in an [FeFe]-hydrogenase that is tuned for H2 oxidation catalysis.

[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays among the [FeFe]-hydrogenases; however, it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of enzymes. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII) exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric electron paramagnetic resonance, we resolved two electron paramagnetic resonance signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 ± 18 mV and -406 ± 12 mV versus normal hydrogen electrode, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.

Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues.

Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5'-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for Cα deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.

Communication between binding sites is required for YqjI regulation of target promoters within the yqjH-yqjI intergenic region.

The nickel-responsive transcription factor YqjI represses its own transcription and transcription of the divergent yqjH gene, which encodes a novel ferric siderophore reductase. The intergenic region between the two promoters is complex, with multiple sequence features that may impact YqjI-dependent regulation of its two target promoters. We utilized mutagenesis and DNase I footprinting to characterize YqjI regulation of the yqjH-yqjI intergenic region. The results show that YqjI binding results in an extended footprint at the yqjI promoter (site II) compared to the yqjH promoter (site I). Mutagenesis of in vivo gene reporter constructs revealed that the two YqjI binding sites, while separated by nearly 200 bp, appear to communicate in order to provide full YqjI-dependent regulation at the two target promoters. Thus, YqjI binding at both promoters is required for full repression of either promoter, suggesting that the two YqjI binding sites cooperate to control transcription from the divergent promoters. Furthermore, internal deletions that shorten the total length of the intergenic region disrupt the ability of YqjI to regulate the yqjH promoter. Finally, mutagenesis of the repetitive extragenic palindromic (REP) elements within the yqjH-yqjI intergenic region shows that these sequences are not required for YqjI regulation. These studies provide a complex picture of novel YqjI transcriptional regulation within the yqjH-yqjI intergenic region and suggest a possible model for communication between the YqjI binding sites at each target promoter.

Fe-S cluster biogenesis by the bacterial Suf pathway.

Biogenesis of iron-sulfur (FeS) clusters in an essential process in living organisms due to the critical role of FeS cluster proteins in myriad cell functions. During biogenesis of FeS clusters, multi-protein complexes are used to drive the mobilization and protection of reactive sulfur and iron intermediates, regulate assembly of various FeS clusters on an ATPase-dependent, multi-protein scaffold, and target nascent clusters to their downstream protein targets. The evolutionarily ancient sulfur formation (Suf) pathway for FeS cluster assembly is found in bacteria and archaea. In Escherichia coli, the Suf pathway functions as an emergency pathway under conditions of iron limitation or oxidative stress. In other pathogenic bacteria, such as Mycobacterium tuberculosis and Enterococcus faecalis, the Suf pathway is the sole source for FeS clusters and therefore is a potential target for the development of novel antibacterial compounds. Here we summarize the considerable progress that has been made in characterizing the first step of mobilization and protection of reactive sulfur carried out by the SufS-SufE or SufS-SufU complex, FeS cluster assembly on SufBC2D scaffold complexes, and the downstream trafficking of nascent FeS clusters to A-type carrier (ATC) proteins. Cell Biology of Metals III edited by Roland Lill and Mick Petris.

Conserved cysteine residues are necessary for nickel-induced allosteric regulation of the metalloregulatory protein YqjI (NfeR) in E. coli.

Transition metal homeostasis is necessary to sustain life. First row transition metals act as cofactors within the cell, performing vital functions ranging from DNA repair to respiration. However, intracellular metal concentrations exceeding physiological requirements may be toxic. In E. coli, the YqjH flavoprotein is thought to play a role in iron homeostasis. YqjH is transcriptionally regulated by the ferric uptake regulator and a newly discovered regulator encoded by yqjI. The apo-form of YqjI is a transcriptional repressor of both the yqjH and yqjI genes. YqjI repressor function is disrupted upon binding of nickel. The YqjI N-terminus is homologous to nickel-binding proteins, implicating this region as a nickel-binding domain. Based on function, yqjI and yqjH should be renamed Ni-responsive Fe-uptake regulator (nfeR) and Ni-responsive Fe-uptake flavoprotein (nfeF), respectively. X-ray Absorption Spectroscopy was employed to characterize the nickel binding site(s) within YqjI. Putative nickel binding ligands were targeted by site-directed mutagenesis and resulting variants were analyzed in vivo for repressor function. Isothermal titration calorimetry and competitive binding assays were used to further quantify nickel interactions with wild-type YqjI and its mutant derivatives. Results indicate plasticity in the nickel binding domain of YqjI. Residues C42 and C43 were found to be required for in vivo response of YqjI to nickel stress, though these residues are not required for in vitro nickel binding. We propose that YqjI may contain a vicinal disulfide bond between C42 and C43 that is important for nickel-responsive allosteric interactions between YqjI domains.