A Van der Waals Interface Hosting Two Groups of Magnetic Skyrmions.

Multiple magnetic skyrmion phases add an additional degree of freedom for skyrmion-based ultrahigh-density spin memory devices. Extending the field to 2D van der Waals magnets is a rewarding challenge, where the realizable degree of freedoms (e.g., thickness, twist angle, and electrical gating) and high skyrmion density result in intriguing new properties and enhanced functionality. In this work, a van der Waals interface, formed by two 2D ferromagnets Cr2 Ge2 Te6 and Fe3 GeTe2 with a Curie temperature of ≈65 and ≈205 K, respectively, hosting two groups of magnetic skyrmions, is reported. Two sets of topological Hall effect signals are observed below 6s0 K when Cr2 Ge2 Te6 is magnetically ordered. These two groups of skyrmions are directly imaged using magnetic force microscopy, and supported by micromagnetic simulations. Interestingly, the magnetic skyrmions persist in the heterostructure with zero applied magnetic field. The results are promising for the realization of skyrmionic devices based on van der Waals heterostructures hosting multiple skyrmion phases.

Combining Superionic Conduction and Favorable Decomposition Products in the Crystalline Lithium-Boron-Sulfur System: A New Mechanism for Stabilizing Solid Li-Ion Electrolytes.

We report a solid-state Li-ion electrolyte predicted to exhibit simultaneously fast ionic conductivity, wide electrochemical stability, low cost, and low mass density. We report exceptional density functional theory (DFT)-based room-temperature single-crystal ionic conductivity values for two phases within the crystalline lithium-boron-sulfur (Li-B-S) system: 62 (+9, -2) mS cm-1 in Li5B7S13 and 80 (-56, -41) mS cm-1 in Li9B19S33. We report significant ionic conductivity values for two additional phases: between 0.0056 and 0.16 mS/cm -1 in Li2B2S5 and between 0.0031 and 9.7 mS cm-1 in Li3BS3 depending on the room-temperature extrapolation scheme used. To our knowledge, our prediction gives Li9B19S33 and Li5B7S13 the second and third highest reported DFT-computed single-crystal ionic conductivities of any crystalline material. We compute the thermodynamic electrochemical stability window widths of these materials to be 0.50 V for Li5B7S13, 0.16 V for Li2B2S5, 0.45 V for Li3BS3, and 0.60 V for Li9B19S33. Individually, these materials exhibit similar or better ionic conductivity and electrochemical stability than the best-known sulfide-based solid-state Li-ion electrolyte materials, including Li10GeP2S12 (LGPS). However, we predict that electrolyte materials synthesized from a range of compositions in the Li-B-S system may exhibit even wider thermodynamic electrochemical stability windows of 0.63 V and possibly as high as 3 V or greater. The Li-B-S system also has a low elemental cost of approximately 0.05 USD/m2 per 10 µm thickness, which is significantly lower than that of germanium-containing LGPS, and a comparable mass density below 2 g/cm3. These fast-conducting phases were initially brought to our attention by a machine learning-based approach to screen over 12,000 solid electrolyte candidates, and the evidence provided here represents an inspiring success for this model.

Characterization of an l-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations.

l-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Here we report that Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses l-ascorbate as sole carbon source via a novel catabolic pathway. RNaseq identified eight candidate catabolic genes, sequence similarity networks, and genome neighborhood networks guided predictions for function of the encoded proteins, and the predictions were confirmed by in vitro assays and in vivo growth phenotypes of gene deletion mutants. l-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b561 and gluconolactonase families, respectively, to form 2,3-diketo-l-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-l-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-l-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD+ catalytically. The product, l-lyxonate, is catabolized to α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.

Coexistence of Magnetic Orders in Two-Dimensional Magnet CrI3.

The magnetic properties in two-dimensional van der Waals materials depend sensitively on structure. CrI3, as an example, has been recently demonstrated to exhibit distinct magnetic properties depending on the layer thickness and stacking order. Bulk CrI3 is ferromagnetic (FM) with a Curie temperature of 61 K and a rhombohedral layer stacking, whereas few-layer CrI3 has a layered antiferromagnetic (AFM) phase with a lower ordering temperature of 45 K and a monoclinic stacking. In this work, we use cryogenic magnetic force microscopy to investigate CrI3 flakes in the intermediate thickness range (25-200 nm) and find that the two types of magnetic orders, hence the stacking orders, can coexist in the same flake with a layer of ∼13 nm at each surface being in the layered AFM phase similar to few-layer CrI3 and the rest in the bulk FM phase. The switching of the bulk moment proceeds through a remnant state with nearly compensated magnetic moment along the c-axis, indicating formation of c-axis domains allowed by a weak interlayer coupling strength in the rhombohedral phase. Our results provide a comprehensive picture on the magnetism in CrI3 and point to the possibility of engineering magnetic heterostructures within the same material.

Using adaptive genetic algorithms combined with high sensitivity single cell-based technology to detect bladder cancer in urine and provide a potential noninvasive marker for response to anti-PD1 immunotherapy.

OBJECTIVE: To use adaptive genetic algorithms (AGA) in combination with single-cell flow cytometry technology to develop a noninvasive test to detect bladder cancer. MATERIALS AND METHODS: Fifty high grade, cystoscopy confirmed, superficial bladder cancer patients, and 15 healthy donor early morning urine samples were collected in an optimized urine collection media. These samples were then used to develop an assay to distinguish healthy from cancer patients' urine using AGA in combination with single-cell flow cytometry technology. Cell recovery and test performance were verified based on cystoscopy and histology for both bladder cancer determination and PD-L1 status. RESULTS: Bladder cancer patients had a significantly higher percentage of white blood cells with substantial PD-L1 expression (P< 0.0001), significantly increased post-G1 epithelial cells (P < 0.005) and a significantly higher DNA index above 1.05 (P < 0.05). AGA allowed parameter optimization to differentiate normal from malignant cells with high accuracy. The resulting prediction model showed 98% sensitivity and 87% specificity with a high area under the ROC value (90%). CONCLUSIONS: Using single-cell technology and machine learning; we developed a new assay to distinguish bladder cancer from healthy patients. Future studies are planned to validate this assay.

Imaging quantum spin Hall edges in monolayer WTe2.

A two-dimensional (2D) topological insulator exhibits the quantum spin Hall (QSH) effect, in which topologically protected conducting channels exist at the sample edges. Experimental signatures of the QSH effect have recently been reported in an atomically thin material, monolayer WTe2. Here, we directly image the local conductivity of monolayer WTe2 using microwave impedance microscopy, establishing beyond doubt that conduction is indeed strongly localized to the physical edges at temperatures up to 77 K and above. The edge conductivity shows no gap as a function of gate voltage, and is suppressed by magnetic field as expected. We observe additional conducting features which can be explained by edge states following boundaries between topologically trivial and nontrivial regions. These observations will be critical for interpreting and improving the properties of devices incorporating WTe2. Meanwhile, they reveal the robustness of the QSH channels and the potential to engineer them in the monolayer material platform.

Functional assignment of multiple catabolic pathways for D-apiose.

Colocation of the genes encoding ABC, TRAP, and TCT transport systems and catabolic pathways for the transported ligand provides a strategy for discovering novel microbial enzymes and pathways. We screened solute-binding proteins (SBPs) for ABC transport systems and identified three that bind D-apiose, a branched pentose in the cell walls of higher plants. Guided by sequence similarity networks (SSNs) and genome neighborhood networks (GNNs), the identities of the SBPs enabled the discovery of four catabolic pathways for D-apiose with eleven previously unknown reactions. The new enzymes include D-apionate oxidoisomerase, which catalyzes hydroxymethyl group migration, as well as 3-oxo-isoapionate-4-phosphate decarboxylase and 3-oxo-isoapionate-4-phosphate transcarboxylase/hydrolase, which are RuBisCO-like proteins (RLPs). The web tools for generating SSNs and GNNs are publicly accessible ( http://efi.igb.illinois.edu/efi-est/ ), so similar 'genomic enzymology' strategies for discovering novel pathways can be used by the community.

Prediction of enzymatic pathways by integrative pathway mapping.

The functions of most proteins are yet to be determined. The function of an enzyme is often defined by its interacting partners, including its substrate and product, and its role in larger metabolic networks. Here, we describe a computational method that predicts the functions of orphan enzymes by organizing them into a linear metabolic pathway. Given candidate enzyme and metabolite pathway members, this aim is achieved by finding those pathways that satisfy structural and network restraints implied by varied input information, including that from virtual screening, chemoinformatics, genomic context analysis, and ligand -binding experiments. We demonstrate this integrative pathway mapping method by predicting the L-gulonate catabolic pathway in Haemophilus influenzae Rd KW20. The prediction was subsequently validated experimentally by enzymology, crystallography, and metabolomics. Integrative pathway mapping by satisfaction of structural and network restraints is extensible to molecular networks in general and thus formally bridges the gap between structural biology and systems biology.

The Metallophore Staphylopine Enables Staphylococcus aureus To Compete with the Host for Zinc and Overcome Nutritional Immunity.

During infection, the host sequesters essential nutrients, such as zinc, to combat invading microbes. Despite the ability of the immune effector protein calprotectin to bind zinc with subpicomolar affinity, Staphylococcus aureus is able to successfully compete with the host for zinc. However, the zinc importers expressed by S. aureus remain unknown. Our investigations have revealed that S. aureus possesses two importers, AdcABC and CntABCDF, which are induced in response to zinc limitation. While AdcABC is similar to known zinc importers in other bacteria, CntABCDF has not previously been associated with zinc acquisition. Concurrent loss of the two systems severely impairs the ability of S. aureus to obtain zinc and grow in zinc-limited environments. Further investigations revealed that the Cnt system is responsible for the ability of S. aureus to compete with calprotectin for zinc in culture and contributes to acquisition of zinc during infection. The cnt locus also enables S. aureus to produce the broad-spectrum metallophore staphylopine. Similarly to the Cnt transporter, loss of staphylopine severely impairs the ability of S. aureus to resist host-imposed zinc starvation, both in culture and during infection. Further investigations revealed that together staphylopine and the Cnt importer function analogously to siderophore-based iron acquisition systems in order to facilitate zinc acquisition by S. aureus Analogous systems are found in a broad range of Gram-positive and Gram-negative bacterial pathogens, suggesting that this new type of zinc importer broadly contributes to the ability of bacteria to cause infection.IMPORTANCE A critical host defense against infection is the restriction of zinc availability. Despite the subpicomolar affinity of the immune effector calprotectin for zinc, Staphylococcus aureus can successfully compete for this essential metal. Here, we describe two zinc importers, AdcABC and CntABCDF, possessed by S. aureus, the latter of which has not previously been associated with zinc acquisition. The ability of S. aureus to compete with the host for zinc is dependent on CntABCDF and the metallophore staphylopine, both in culture and during infection. These results expand the mechanisms utilized by bacteria to obtain zinc, beyond Adc-like systems, and demonstrate that pathogens utilize strategies similar to siderophore-based iron acquisition to obtain other essential metals during infection. The staphylopine synthesis machinery is present in a diverse collection of bacteria, suggesting that this new family of zinc importers broadly contributes to the ability of numerous pathogens to cause infection.

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars.

Using a large-scale "genomic enzymology" approach, we (i) assigned novel ATP-dependent four-carbon acid sugar kinase functions to members of the DUF1537 protein family (domain of unknown function; Pfam families PF07005 and PF17042) and (ii) discovered novel catabolic pathways for d-threonate, l-threonate, and d-erythronate. The experimentally determined ligand specificities of several solute binding proteins (SBPs) for TRAP (tripartite ATP-independent permease) transporters for four-carbon acids, including d-erythronate and l-erythronate, were used to constrain the substrates for the catabolic pathways that degrade the SBP ligands to intermediates in central carbon metabolism. Sequence similarity networks and genome neighborhood networks were used to identify the enzyme components of the pathways. Conserved genome neighborhoods encoded SBPs as well as permease components of the TRAP transporters, members of the DUF1537 family, and a member of the 4-hydroxy-l-threonine 4-phosphate dehydrogenase (PdxA) oxidative decarboxylase, class II aldolase, or ribulose 1,5-bisphosphate carboxylase/oxygenase, large subunit (RuBisCO) superfamily. Because the characterized substrates of members of the PdxA, class II aldolase, and RuBisCO superfamilies are phosphorylated, we postulated that the members of the DUF1537 family are novel ATP-dependent kinases that participate in catabolic pathways for four-carbon acid sugars. We determined that (i) the DUF1537/PdxA pair participates in a pathway for the conversion of d-threonate to dihydroxyacetone phosphate and CO2 and (ii) the DUF1537/class II aldolase pair participates in pathways for the conversion of d-erythronate and l-threonate (epimers at carbon-3) to dihydroxyacetone phosphate and CO2 The physiological importance of these pathways was demonstrated in vivo by phenotypic and genetic analyses.

Mitochondrial cytochrome c biogenesis: no longer an enigma.

Cytochromes c (cyt c) and c1 are heme proteins that are essential for aerobic respiration. Release of cyt c from mitochondria is an important signal in apoptosis initiation. Biogenesis of c-type cytochromes involves covalent attachment of heme to two cysteines (at a conserved CXXCH sequence) in the apocytochrome. Heme attachment is catalyzed in most mitochondria by holocytochrome c synthase (HCCS), which is also necessary for the import of apocytochrome c (apocyt c). Thus, HCCS affects cellular levels of cyt c, impacting mitochondrial physiology and cell death. Here, we review the mechanisms of HCCS function and the roles of heme and residues in the CXXCH motif. Additionally, we consider concepts emerging within the two prokaryotic cytochrome c biogenesis pathways.

Experimental strategies for functional annotation and metabolism discovery: targeted screening of solute binding proteins and unbiased panning of metabolomes.

The rate at which genome sequencing data is accruing demands enhanced methods for functional annotation and metabolism discovery. Solute binding proteins (SBPs) facilitate the transport of the first reactant in a metabolic pathway, thereby constraining the regions of chemical space and the chemistries that must be considered for pathway reconstruction. We describe high-throughput protein production and differential scanning fluorimetry platforms, which enabled the screening of 158 SBPs against a 189 component library specifically tailored for this class of proteins. Like all screening efforts, this approach is limited by the practical constraints imposed by construction of the library, i.e., we can study only those metabolites that are known to exist and which can be made in sufficient quantities for experimentation. To move beyond these inherent limitations, we illustrate the promise of crystallographic- and mass spectrometric-based approaches for the unbiased use of entire metabolomes as screening libraries. Together, our approaches identified 40 new SBP ligands, generated experiment-based annotations for 2084 SBPs in 71 isofunctional clusters, and defined numerous metabolic pathways, including novel catabolic pathways for the utilization of ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an integrated strategy for realizing the full value of amassing genome sequence data.

Mechanisms of mitochondrial holocytochrome c synthase and the key roles played by cysteines and histidine of the heme attachment site, Cys-XX-Cys-His.

Mitochondrial cytochrome c assembly requires the covalent attachment of heme by thioether bonds between heme vinyl groups and a conserved CXXCH motif of cytochrome c/c1. The enzyme holocytochrome c synthase (HCCS) binds heme and apocytochrome c substrate to catalyze this attachment, subsequently releasing holocytochrome c for proper folding to its native structure. We address mechanisms of assembly using a functional Escherichia coli recombinant system expressing human HCCS. Human cytochrome c variants with individual cysteine, histidine, double cysteine, and triple cysteine/histidine substitutions (of CXXCH) were co-purified with HCCS. Single and double mutants form a complex with HCCS but not the triple mutant. Resonance Raman and UV-visible spectroscopy support the proposal that heme puckering induced by both thioether bonds facilitate release of holocytochrome c from the complex. His-19 (of CXXCH) supplies the second axial ligand to heme in the complex, the first axial ligand was previously shown to be from HCCS residue His-154. Substitutions of His-19 in cytochrome c to seven other residues (Gly, Ala, Met, Arg, Lys, Cys, and Tyr) were used with various approaches to establish other roles played by His-19. Three roles for His-19 in HCCS-mediated assembly are suggested: (i) to provide the second axial ligand to the heme iron in preparation for covalent attachment; (ii) to spatially position the two cysteinyl sulfurs adjacent to the two heme vinyl groups for thioether formation; and (iii) to aid in release of the holocytochrome c from the HCCS active site. Only H19M is able to carry out these three roles, albeit at lower efficiencies than the natural His-19.

Conserved residues of the human mitochondrial holocytochrome c synthase mediate interactions with heme.

C-type cytochromes are distinguished by the covalent attachment of a heme cofactor, a modification that is typically required for its subsequent folding, stability, and function. Heme attachment takes place in the mitochondrial intermembrane space and, in most eukaryotes, is mediated by holocytochrome c synthase (HCCS). HCCS is the primary component of the eukaryotic cytochrome c biogenesis pathway, known as System III. The catalytic function of HCCS depends on its ability to coordinate interactions between its substrates: heme and cytochrome c. Recent advancements in the recombinant expression and purification of HCCS have facilitated comprehensive analyses of the roles of conserved residues in HCCS, as demonstrated in this study. Previously, we proposed a four-step model describing HCCS-mediated cytochrome c assembly, identifying a conserved histidine residue (His154) as an axial ligand to the heme iron. In this study, we performed a systematic mutational analysis of 17 conserved residues in HCCS, and we provide evidence that the enzyme contains two heme-binding domains. Our data indicate that heme contacts mediated by residues within these domains modulate the dynamics of heme binding and contribute to the stability of the HCCS-heme-cytochrome c steady state ternary complex. While some residues are essential for initial heme binding (step 1), others impact the subsequent release of the holocytochrome c product (step 4). Certain HCCS mutants that were defective in heme binding were corrected for function by exogenous aminolevulinic acid (ALA, the precursor to heme). This chemical "correction" supports the proposed role of heme binding for the corresponding residues.

Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks.

Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ∼85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

Interaction of holoCcmE with CcmF in heme trafficking and cytochrome c biosynthesis.

The periplasmic heme chaperone holoCcmE is essential for heme trafficking in the cytochrome c biosynthetic pathway in many bacteria, archaea, and plant mitochondria. This pathway, called system I, involves two steps: (i) formation and release of holoCcmE (by the ABC-transporter complex CcmABCD) and (ii) delivery of the heme in holoCcmE to the putative cytochrome c heme lyase complex, CcmFH. CcmFH is believed to facilitate the final covalent attachment of heme (from holoCcmE) to the apocytochrome c. Although most models for system I propose that holoCcmE delivers heme directly to CcmF, no interaction between holoCcmE and CcmF has been demonstrated. Here, a complex between holoCcmE and CcmF is "trapped", purified, and characterized. HoloCcmE must be released from the ABC-transporter complex CcmABCD to interact with CcmF, and the holo-form of CcmE interacts with CcmF at levels at least 20-fold higher than apoCcmE. Two conserved histidines (here termed P-His1 and P-His2) in separate periplasmic loops in CcmF are required for interaction with holoCcmE, and evidence that P-His1 and P-His2 function as heme-binding ligands is presented. These results show that heme in holoCcmE is essential for complex formation with CcmF and that the heme of holoCcmE is coordinated by P-His1 and P-His2 within the WWD domain of CcmF. These features are strikingly similar to formation of the CcmC:heme:CcmE ternary complex [Richard-Fogal C, Kranz RG. The CcmC:heme:CcmE complex in heme trafficking and cytochrome c biosynthesis. J Mol Biol 2010;401:350­62] and suggest common mechanistic and structural aspects.

The CcmFH complex is the system I holocytochrome c synthetase: engineering cytochrome c maturation independent of CcmABCDE.

Cytochrome c maturation (ccm) in many bacteria, archaea and plant mitochondria requires eight membrane proteins, CcmABCDEFGH, called system I. This pathway delivers and attaches haem covalently to two cysteines (of Cys-Xxx-Xxx-Cys-His) in the cytochrome c. All models propose that CcmFH facilitates covalent attachment of haem to the apocytochrome; namely, that it is the synthetase. However, holocytochrome c synthetase activity has not been directly demonstrated for CcmFH. We report formation of holocytochromes c by CcmFH and CcmG, a periplasmic thioredoxin, independent of CcmABCDE (we term this activity CcmFGH-only). Cytochrome c produced in the absence of CcmABCDE is indistinguishable from cytochrome c produced by the full system I, with a cleaved signal sequence and two covalent bonds to haem. We engineered increased cytochrome c production by CcmFGH-only, with yields approaching those from the full system I. Three conserved histidines in CcmF (TM-His1, TM-His2 and P-His1) are required for activity, as are the conserved cysteine pairs in CcmG and CcmH. Our findings establish that CcmFH is the system I holocytochrome c synthetase. Although we discuss why this engineering would likely not replace the need for CcmABCDE in nature, these results provide unique mechanistic and evolutionary insights into cytochrome c biosynthesis.

Human mitochondrial holocytochrome c synthase's heme binding, maturation determinants, and complex formation with cytochrome c.

Proper functioning of the mitochondrion requires the orchestrated assembly of respiratory complexes with their cofactors. Cytochrome c, an essential electron carrier in mitochondria and a critical component of the apoptotic pathway, contains a heme cofactor covalently attached to the protein at a conserved CXXCH motif. Although it has been known for more than two decades that heme attachment requires the mitochondrial protein holocytochrome c synthase (HCCS), the mechanism remained unknown. We purified membrane-bound human HCCS with endogenous heme and in complex with its cognate human apocytochrome c. Spectroscopic analyses of HCCS alone and complexes of HCCS with site-directed variants of cytochrome c revealed the fundamental steps of heme attachment and maturation. A conserved histidine in HCCS (His154) provided the key ligand to the heme iron. Formation of the HCCS:heme complex served as the platform for interaction with apocytochrome c. Heme was the central molecule mediating contact between HCCS and apocytochrome c. A conserved histidine in apocytochrome c (His19 of CXXCH) supplied the second axial ligand to heme in the trapped HCCS:heme:cytochrome c complex. We also examined the substrate specificity of human HCCS and converted a bacterial cytochrome c into a robust substrate for the HCCS. The results allow us to describe the molecular mechanisms underlying the HCCS reaction.

Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III.

The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Heme ligand identification and redox properties of the cytochrome c synthetase, CcmF.

Cytochrome c maturation in many bacteria, archaea, and plant mitochondria involves the integral membrane protein CcmF, which is thought to function as a cytochrome c synthetase by facilitating the final covalent attachment of heme to the apocytochrome c. We previously reported that the E. coli CcmF protein contains a b-type heme that is stably and stoichiometrically associated with the protein and is not the heme attached to apocytochrome c. Here, we show that mutation of either of two conserved transmembrane histidines (His261 or His491) impairs stoichiometric b-heme binding in CcmF and results in spectral perturbations in the remaining heme. Exogeneous imidazole is able to correct cytochrome c maturation for His261 and His491 substitutions with small side chains (Ala or Gly), suggesting that a "cavity" is formed in these CcmF mutants in which imidazole binds and acts as a functional ligand to the b-heme. The results of resonance Raman spectroscopy on wild-type CcmF are consistent with a hexacoordinate low-spin b-heme with at least one endogeneous axial His ligand. Analysis of purified recombinant CcmF proteins from diverse prokaryotes reveals that the b-heme in CcmF is widely conserved. We have also determined the reduction potential of the CcmF b-heme (E(m,7) = -147 mV). We discuss these results in the context of CcmF structure and functions as a heme reductase and cytochrome c synthetase.