A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Aspartate or arginine? Validated redox state X-ray structures elucidate mechanistic subtleties of FeIV = O formation in bacterial dye-decolorizing peroxidases.

Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2 O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+ , was exposed to O2 or H2 O2 . We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2 O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O2 . Initially formed Fe3+ can further react with H2 O2 (producing protein bound radicals) but relaxes within seconds to an H2 O2 -unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2 O2 rather than sequester iron.

Serial Femtosecond Zero Dose Crystallography Captures a Water-Free Distal Heme Site in a Dye-Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in FeIV =O Formation.

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.

A Histidine Residue and a Tetranuclear Cuprous-thiolate Cluster Dominate the Copper Loading Landscape of a Copper Storage Protein from Streptomyces lividans.

The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high-affinity binding sites that use cuprous-thiolate coordination chemistry. In bacteria, a family of cysteine rich four-helix bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four-helix bundle, facilitated by a set of three histidine residues. X-ray crystallography reveals the existence of polynuclear cuprous-thiolate clusters culminating in the assembly of a tetranuclear [Cu4 (µ2 -S-Cys)4 (Νδ1 -His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous-thiolate clusters as observed in the outer core. Using site-directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed.

An Aromatic Dyad Motif in Dye Decolourising Peroxidases Has Implications for Free Radical Formation and Catalysis.

Dye decolouring peroxidases (DyPs) are the most recent class of heme peroxidase to be discovered. On reacting with H2 O2 , DyPs form a high-valent iron(IV)-oxo species and a porphyrin radical (Compound I) followed by stepwise oxidation of an organic substrate. In the absence of substrate, the ferryl species decays to form transient protein-bound radicals on redox active amino acids. Identification of radical sites in DyPs has implications for their oxidative mechanism with substrate. Using a DyP from Streptomyces lividans, referred to as DtpA, which displays low reactivity towards synthetic dyes, activation with H2 O2 was explored. A Compound I EPR spectrum was detected, which in the absence of substrate decays to a protein-bound radical EPR signal. Using a newly developed version of the Tyrosyl Radical Spectra Simulation Algorithm, the radical EPR signal was shown to arise from a pristine tyrosyl radical and not a mixed Trp/Tyr radical that has been widely reported in DyP members exhibiting high activity with synthetic dyes. The radical site was identified as Tyr374, with kinetic studies inferring that although Tyr374 is not on the electron-transfer pathway from the dye RB19, its replacement with a Phe does severely compromise activity with other organic substrates. These findings hint at the possibility that alternative electron-transfer pathways for substrate oxidation are operative within the DyP family. In this context, a role for a highly conserved aromatic dyad motif is discussed.

Naturally Occurring Disease-Related Mutations in the 40-57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization.

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p Ktrigger). A second deprotonation stabilizes a pentacoordinate heme form (p Ka2). In this study, site-directed variants at the 41 and 48 positions in the 40-57 Ω-loop and at the 81 and 83 positions in the 71-85 Ω-loop reveal that conformational transitions in the 71-85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40-57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the p Ktrigger and increase p Ka2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80-Fe(III) bond strength.

Active-site maturation and activity of the copper-radical oxidase GlxA are governed by a tryptophan residue.

GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best characterised. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic sites in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper-co-ordinating tyrosine ligand and houses a radical. In GlxA and Gox, a second co-ordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes, creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here, we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.

Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome c Increases Its Peroxidatic Activity.

Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. The Y48H variant arises from one such mutation and is found in the 40-57 Ω-loop, the lowest-unfolding free energy substructure of the cytochrome c fold. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes compared to the wild-type structure, with the axial Met80 ligand coordinated to the heme iron. Despite this, the intrinsic peroxidase activity is enhanced, implying that a pentacoordinate heme state is more prevalent in the Y48H variant, corroborated through determination of a Met80 "off rate" of >125 s-1 compared to a rate of ∼6 s-1 for the wild-type protein. Heteronuclear nuclear magnetic resonance measurements with the oxidized Y48H variant reveal heightened dynamics in the 40-57 Ω-loop and the Met80-containing 71-85 Ω-loop relative to the wild-type protein, illustrating communication between these substructures. Placed into context with the G41S cytochrome c variant, also implicated in thrombocytopenia, a dynamic picture associated with this disease relative to cytochrome c is emerging whereby increasing dynamics in substructures of the cytochrome c fold serve to facilitate an increased population of the peroxidatic pentacoordinate heme state in the following order: wild type < G41S < Y48H.

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases.

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.

Tyrosine or Tryptophan? Modifying a Metalloradical Catalytic Site by Removal of the Cys-Tyr Cross-Link in the Galactose 6-Oxidase Homologue GlxA.

The concerted redox action of a metal ion and an organic cofactor is a unique way to maximize the catalytic power of an enzyme. An example of such synergy is the fungal galactose 6-oxidase, which has inspired the creation of biomimetic copper oxidation catalysts. Galactose 6-oxidase and its bacterial homologue, GlxA, possess a metalloradical catalytic site that contains a free radical on a covalently linked Cys-Tyr and a copper atom. Such a catalytic site enables for the two-electron oxidation of alcohols to aldehydes. When the ability to form the Cys-Tyr in GlxA is disrupted, a radical can still be formed. Surprisingly, the radical species is not the Tyr residue but rather a copper second-coordination sphere Trp residue. This is demonstrated through the introduction of a new algorithm for Trp-radical EPR spectra simulation. Our findings suggest a new mechanism of free-radical transfer between aromatic residues and that the Cys-Tyr cross-link prevents radical migration away from the catalytic site.

GlxA is a new structural member of the radical copper oxidase family and is required for glycan deposition at hyphal tips and morphogenesis of Streptomyces lividans.

Streptomyces lividans displays a distinct dependence on copper to fully initiate morphological development. Evidence has accumulated to implicate the participation of an extracytoplasmic cuproenzyme in morphogenesis. In the present study, we show that GlxA fulfils all criteria to be that cuproenzyme. GlxA is membrane associated and has an active site consisting of a mononuclear copper and a cross-linked Y-C cofactor. The domain organization of the tertiary structure defines GlxA as a new structural member of the mono-copper oxidase family, with copper co-ordination geometry similar to, but spectroscopically distinct from fungal galactose oxidase (Gox). EPR spectroscopy reveals that the oxidation of cupric GlxA generates a protein radical residing on the Y-C cross-link. A variety of canonical Gox substrates (including D-galactose) were tested but none were readily turned over by GlxA. A glxA null-mutant leads to loss of glycan accumulation at hyphal tips and consequently a drastically changed morphology both on solid substrates and in liquid-grown environments, a scenario similarly observed in the absence of the neighbouring glycan synthase CslA (cellulase synthase-like protein). In addition the glxA mutant has lost the stimulation of development by copper, supporting a model whereby the enzymatic action of GlxA on the glycan is required for development and morphology. From a biotechnology perspective, the open mycelium morphology observed with the glxA mutant in submerged culture has implications for use as an enzyme production host.

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans.

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR(Sl)) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR(Sl) binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5'-TAC/GTA-3' inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR(Sl)-DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoR(Sl) tetramers. Finally, differential binding modes may exist in operator sites having more than one 5'-TAC/GTA-3' inverted repeat with implications in vivo for a mechanism of modular control.

Structural insights into conformational switching in the copper metalloregulator CsoR from Streptomyces lividans.

Copper-sensitive operon repressors (CsoRs) act to sense cuprous ions and bind them with a high affinity under copper stress in many bacteria. The binding of copper(I) leads to a conformational change in their homotetramer structure, causing disassembly of the operator DNA-CsoR complex and evoking a transcriptional response. Atomic-level structural insight into the conformational switching mechanism between the apo and metal-bound states is lacking. Here, a new X-ray crystal structure of the CsoR from Streptomyces lividans is reported and compared with a previously reported S. lividans CsoR X-ray structure crystallized under different conditions. Based on evidence from this new X-ray structure, it is revealed that the conformational switching between states centres on a concertina effect at the C-terminal end of each α2 helix in the homotetramer. This drives the Cys104 side chain, a copper(I)-ligating residue, into a position enabling copper(I) coordination and as a result disrupts the α2-helix geometry, leading to a compacting and twisting of the homotetramer structure. Strikingly, the conformational switching induces a redistribution of electrostatic surface potential on the tetrameric DNA-binding face, which in the copper(I)-bound state would no longer favour interaction with the mode of operator DNA binding.

Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans.

In Streptomyces lividans an extracytoplasmic copper-binding Sco protein plays a role in two unlinked processes: (i) initiating a morphological development switch and (ii) facilitating the co-factoring of the CuA domain of CcO (cytochrome c oxidase). How Sco obtains copper once secreted to the extracytoplasmic environment is unknown. In the present paper we report on a protein possessing an HX6MX21HXM motif that binds a single cuprous ion with subfemtomolar affinity. High-resolution X-ray structures of this extracytoplasmic copper chaperone-like protein (ECuC) in the apo- and Cu(I)-bound states reveal that the latter possesses a surface-accessible cuprous-ion-binding site located in a dish-shaped region of ß-sheet structure. A cuprous ion is transferred under a favourable thermodynamic gradient from ECuC to Sco with no back transfer occurring. The ionization properties of the cysteine residues in the Cys86xxxCys9° copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys86 in initiating an inter-complex ligand-exchange reaction with Cu(I)-ECuC. Generation of the genetic knockouts, Δsco, Δecuc and Δsco/ecuc, and subsequent in vivo assays lend support to the existence of a branched extracytoplasmic copper-trafficking pathway in S. lividans. One branch requires both Sco and to a certain extent ECuC to cofactor the CuA domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.

The hydrogen-peroxide-induced radical behaviour in human cytochrome c-phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant.

We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met(80) ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr(46) and Tyr(48), have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.

Efficient in situ screening of and data collection from microcrystals in crystallization plates.

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

The oligomeric states of dye-decolorizing peroxidases from Streptomyces lividans and their implications for mechanism of substrate oxidation.

A common evolutionary mechanism in biology to drive function is protein oligomerization. In prokaryotes, the symmetrical assembly of repeating protein units to form homomers is widespread, yet consideration in vitro of whether such assemblies have functional or mechanistic consequences is often overlooked. Dye-decolorizing peroxidases (DyPs) are one such example, where their dimeric α + ß barrel units can form various oligomeric states, but the oligomer influence, if any, on mechanism and function has received little attention. In this work, we have explored the oligomeric state of three DyPs found in Streptomyces lividans, each with very different mechanistic behaviors in their reactions with hydrogen peroxide and organic substrates. Using analytical ultracentrifugation, we reveal that except for one of the A-type DyPs where only a single sedimenting species is detected, oligomer states ranging from homodimers to dodecamers are prevalent in solution. Using cryo-EM on preparations of the B-type DyP, we determined a 3.02 Å resolution structure of a hexamer assembly that corresponds to the dominant oligomeric state in solution as determined by analytical ultracentrifugation. Furthermore, cryo-EM data detected sub-populations of higher-order oligomers, with one of these formed by an arrangement of two B-type DyP hexamers to give a dodecamer assembly. Our solution and structural insights of these oligomer states provide a new framework to consider previous mechanistic studies of these DyP members and are discussed in terms of long-range electron transfer for substrate oxidation and in the "storage" of oxidizable equivalents on the heme until a two-electron donor is available.

Response to copper stress in Streptomyces lividans extends beyond genes under direct control of a copper-sensitive operon repressor protein (CsoR).

A copper-sensitive operon repressor protein (CsoR) has been identified in Streptomyces lividans (CsoR(Sl)) and found to regulate copper homeostasis with attomolar affinity for Cu(I). Solution studies reveal apo- and Cu(I)-CsoR(Sl) to be a tetramer assembly, and a 1.7-Å resolution crystal structure of apo-CsoR(Sl) reveals that a significant conformational change is necessary to enable Cu(I) binding. In silico prediction of the CsoR regulon was confirmed in vitro (EMSA) and in vivo (RNA-seq), which highlighted that next to the csoR gene itself, the regulon consists of two Cu(I) efflux systems involving a CopZ-like copper metallochaperone protein and a CopA P(1)-type ATPase. Although deletion of csoR has only minor effects on S. lividans development when grown under high copper concentrations, mutations of the Cu(I) ligands decrease tolerance to copper as a result of the Cu(I)-CsoR mutants failing to disengage from the DNA targets, thus inhibiting the derepression of the regulon. RNA-seq experiments carried out on samples incubated with exogenous copper and a ΔcsoR strain showed that the set of genes responding to copper stress is much wider than anticipated and largely extends beyond genes targeted by CsoR. This suggests more control levels are operating and directing other regulons in copper homeostasis beside the CsoR regulon.

An investigation into a cardiolipin acyl chain insertion site in cytochrome c.

Mitochondrial cytochrome c associates with the phosphoplipid cardiolipin (CL) through a combination of electrostatic and hydrophobic interactions. The latter occurs by insertion into cytochrome c of an acyl chain, resulting in the dissociation of the axial Met-80 heme-iron ligand. The resulting five coordinate cytochrome c/CL complex has peroxidatic properties leading to peroxidation of CL and dissociation of the complex. These events are considered to be pre-apoptotic and culminate with release of cytochrome c from the mitochondria into the cytoplasm. Two distinct surface regions on cytochrome c have been suggested to mediate CL acyl chain insertion and this study has probed one of these regions. We have constructed a series of alanine mutants aimed at disrupting a surface cleft formed between residues 67-71 and 82-85. The physicochemical properties, peroxidase activity, CL binding, and kinetics of carbon monoxide (CO) binding to the ferrous cytochrome c/CL complex have been assessed for the individual mutants. Our findings reveal that the majority of mutants are capable of binding CL in the same apparent stoichiometry as the wild-type protein, with the extent to which the Met-80 ligand is bound in the ferrous cytochrome c/CL complex being mutant specific at neutral pH. Mutation of the species conserved Arg-91 residue, that anchors the cleft, results in the greatest changes to physicochemical properties of the protein leading to a change in the CL binding ratio required to effect structural changes and to the ligand-exchange properties of the ferrous cytochrome c/CL complex.