RESUMO
In Gram-negative bacteria, outer-membrane lipoproteins are essential for maintaining cellular integrity, transporting nutrients, establishing infections, and promoting the formation of biofilms. The LolCDE ABC transporter, LolA chaperone, and LolB outer-membrane receptor form an essential system for transporting newly matured lipoproteins from the outer leaflet of the cytoplasmic membrane to the innermost leaflet of the outer membrane. Here, we present a crystal structure of LolA in complex with the periplasmic domain of LolC. The structure reveals how a solvent-exposed ß-hairpin loop (termed the "Hook") and trio of surface residues (the "Pad") of LolC are essential for recruiting LolA from the periplasm and priming it to receive lipoproteins. Experiments with purified LolCDE complex demonstrate that association with LolA is independent of nucleotide binding and hydrolysis, and homology models based on the MacB ABC transporter predict that LolA recruitment takes place at a periplasmic site located at least 50 Å from the inner membrane. Implications for the mechanism of lipoprotein extraction and transfer are discussed. The LolA-LolC structure provides atomic details on a key protein interaction within the Lol pathway and constitutes a vital step toward the complete molecular understanding of this important system.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Periplasma/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/química , Proteínas de Escherichia coli/química , Hidrólise , Modelos Moleculares , Proteínas Periplásmicas de Ligação/química , Mapeamento de Interação de Proteínas , Transporte ProteicoRESUMO
MacB is an ABC transporter that collaborates with the MacA adaptor protein and TolC exit duct to drive efflux of antibiotics and enterotoxin STII out of the bacterial cell. Here we present the structure of ATP-bound MacB and reveal precise molecular details of its mechanism. The MacB transmembrane domain lacks a central cavity through which substrates could be passed, but instead conveys conformational changes from one side of the membrane to the other, a process we term mechanotransmission. Comparison of ATP-bound and nucleotide-free states reveals how reversible dimerization of the nucleotide binding domains drives opening and closing of the MacB periplasmic domains via concerted movements of the second transmembrane segment and major coupling helix. We propose that the assembled tripartite pump acts as a molecular bellows to propel substrates through the TolC exit duct, driven by MacB mechanotransmission. Homologs of MacB that do not form tripartite pumps, but share structural features underpinning mechanotransmission, include the LolCDE lipoprotein trafficking complex and FtsEX cell division signaling protein. The MacB architecture serves as the blueprint for understanding the structure and mechanism of an entire ABC transporter superfamily and the many diverse functions it supports.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/química , Aggregatibacter actinomycetemcomitans/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Aggregatibacter actinomycetemcomitans/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sítios de Ligação , Transporte Biológico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, leading to stabilization of various cullin-RING ligase targets, and also prevents polyubiquitin chain formation. Here, we report the crystal structures of two Cif/NEDD8 complexes, revealing a conserved molecular interface that defines enzyme/substrate recognition. Mutation of residues forming the interface suggests that shape complementarity, rather than specific individual interactions, is a critical feature for complex formation. We show that Cifs from diverse bacteria bind NEDD8 in vitro and conclude that they will all interact with their substrates in the same way. The "occluding loop" in Cif gates access to Gln40 by forcing a conformational change in the C terminus of NEDD8. We used native PAGE to follow the activity of Cif from the human pathogen Yersinia pseudotuberculosis and selected variants, and the position of Gln40 in the active site has allowed us to propose a catalytic mechanism for these enzymes.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Photorhabdus/enzimologia , Ubiquitinas/química , Ubiquitinas/metabolismo , Yersinia pseudotuberculosis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalização , Glutamina/genética , Células HeLa , Interações Hospedeiro-Parasita/fisiologia , Humanos , Dados de Sequência Molecular , Mutagênese/fisiologia , Proteína NEDD8 , Proteína Oncogênica p21(ras)/metabolismo , Photorhabdus/genética , Poliubiquitina/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Ubiquitinas/genética , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Yersinia pseudotuberculosis/genética , Infecções por Yersinia pseudotuberculosis/metabolismo , Infecções por Yersinia pseudotuberculosis/microbiologiaRESUMO
Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram-positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-visible spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, which are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Although current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.
Assuntos
Adesinas Bacterianas/química , Ésteres/química , Fímbrias Bacterianas/metabolismo , Streptococcus pyogenes/metabolismo , Alelos , Ácido Aspártico/química , Adesão Celular , Cristalografia por Raios X/métodos , Escherichia coli/metabolismo , Lisina/química , Mutagênese , Peptídeos/química , Ligação Proteica , Espectrofotometria Ultravioleta/métodos , Streptococcus pneumoniae/metabolismoRESUMO
BACKGROUND: To satisfy their requirement for iron while at the same time countering the toxicity of this highly reactive metal ion, prokaryotes have evolved proteins belonging to two distinct sub-families of the ferritin family: the bacterioferritins (BFRs) and the bacterial ferritins (Ftns). Recently, Ftn homologues have also been identified and characterised in archaeon species. All of these prokaryotic ferritins function by solubilising and storing large amounts of iron in the form of a safe but bio-available mineral. SCOPE OF REVIEW: The mechanism(s) by which the iron mineral is formed by these proteins is the subject of much current interest. Here we review the available information on these proteins, with particular emphasis on significant advances resulting from recent structural, spectroscopic and kinetic studies. MAJOR CONCLUSIONS: Current understanding indicates that at least two distinct mechanisms are in operation in prokaryotic ferritins. In one, the ferroxidase centre acts as a true catalytic centre in driving Fe(2+) oxidation in the cavity; in the other, the centre acts as a gated iron pore by oxidising Fe(2+) and transferring the resulting Fe(3+) into the central cavity. GENERAL SIGNIFICANCE: The prokaryotic ferritins exhibit a wide variation in mechanisms of iron core mineralisation. The basis of these differences lies, at least in part, in structural differences at and around the catalytic centre. However, it appears that more subtle differences must also be important in controlling the iron chemistry of these remarkable proteins.
Assuntos
Ferritinas/química , Ferritinas/metabolismo , Ferro/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Compostos Férricos/química , Compostos Férricos/metabolismo , Ferro/química , Minerais/química , Minerais/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Células Procarióticas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Ferritins solubilize and detoxify the essential metal iron through formation of a ferric mineral within the protein's central cavity. Key to this activity is an intrasubunit catalytic dinuclear iron center called the ferroxidase center. Here we show that the fluorescence intensity of Escherichia coli bacterioferritin (BFR), due to the presence of two tryptophan residues (Trp35 and Trp133) in each of the 24 subunits, is highly sensitive to the iron status of the ferroxidase center and is quenched to different extents by Fe2+ and Fe3+. Recovery of the quench following oxidation of Fe2+ to Fe3+ at the ferroxidase center was not observed, indicating that the di-Fe3+ form of the center is stable. Studies of the single-tryptophan variants W35F and W133F showed that Trp133, which lies approximately 10 A from the ferroxidase center, is primarily responsible for the observed fluorescence sensitivity to iron, while studies of a stable E. coli BFR subunit dimer demonstrated that the observed quench properties are principally derived from the interaction of iron with tryptophan residues within the subunit dimer. A double-tryptophan variant (W35F/W133F) was found to exhibit fluorescence from the seven tyrosine residues present in each subunit, which was also sensitive to the iron status of the ferroxidase center. Finally, we demonstrate using Zn2+, a potent competitive inhibitor of Fe2+ binding and oxidation, that the fluorescence response can be used to monitor the loss of iron from the ferroxidase center.
Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos b/química , Proteínas de Escherichia coli/química , Ferritinas/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/metabolismo , Primers do DNA/genética , Dimerização , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferritinas/genética , Ferritinas/metabolismo , Ferro/química , Ferro/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca(2+), at an inter-domain position some 14 A away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as -75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK(a), consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca(2+)-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca(2+)-depleted BdbD was significantly less stable than reduced Ca(2+)-containing protein, and the midpoint reduction potential was shifted by approximately -20 mV, suggesting that Ca(2+) functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.
Assuntos
Proteínas de Bactérias/química , Cálcio/química , Proteína Dissulfeto Redutase (Glutationa)/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Cristalização , Conformação Molecular , Dados de Sequência Molecular , Oxirredução , Ligação Proteica , Proteína Dissulfeto Redutase (Glutationa)/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a gamma-proteobacterium) and human pathogen Burkholderia pseudomallei (a beta-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the 'tail-domain' (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.
Assuntos
Proteínas de Bactérias/química , Burkholderia pseudomallei/química , Photorhabdus/química , Sequência de Aminoácidos , Burkholderia pseudomallei/metabolismo , Domínio Catalítico , Cromatografia em Gel , Cristalografia por Raios X , Dados de Sequência Molecular , Photorhabdus/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Ferritin proteins function to detoxify, solubilize and store cellular iron by directing the synthesis of a ferric oxyhydroxide mineral solubilized within the protein's central cavity. Here, through the application of X-ray crystallographic and kinetic methods, we report significant new insight into the mechanism of mineralization in a bacterioferritin (BFR). The structures of nonheme iron-free and di-Fe(2+) forms of BFR showed that the intrasubunit catalytic center, known as the ferroxidase center, is preformed, ready to accept Fe(2+) ions with little or no reorganization. Oxidation of the di-Fe(2+) center resulted in a di-Fe(3+) center, with bridging electron density consistent with a mu-oxo or hydro bridged species. The mu-oxo bridged di-Fe(3+) center appears to be stable, and there is no evidence that Fe(3+)species are transferred into the core from the ferroxidase center. Most significantly, the data also revealed a novel Fe(2+) binding site on the inner surface of the protein, lying approximately 10 A directly below the ferroxidase center, coordinated by only two residues, His46 and Asp50. Kinetic studies of variants containing substitutions of these residues showed that the site is functionally important. In combination, the data support a model in which the ferroxidase center functions as a true catalytic cofactor, rather than as a pore for the transfer of iron into the central cavity, as found for eukaryotic ferritins. The inner surface iron site appears to be important for the transfer of electrons, derived from Fe(2+) oxidation in the cavity, to the ferroxidase center. Bacterioferritin may represent an evolutionary link between ferritins and class II di-iron proteins not involved in iron metabolism.
Assuntos
Proteínas de Bactérias/química , Ceruloplasmina/química , Grupo dos Citocromos b/química , Ferritinas/química , Ferro/química , Minerais/química , Cristalografia por Raios X , Evolução Molecular , Oxirredução , Conformação ProteicaRESUMO
Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens express a "housekeeping" sortase that recognizes the majority of secreted proteins containing an LPXTG wall-sorting motif and covalently attaches these to bacterial cell wall peptidoglycan. Many Gram-positive pathogens also express additional sortases that link a small number of proteins, often with variant wall-sorting motifs, to either other surface proteins or peptidoglycan. To better understand the mechanisms of catalysis and substrate recognition by the housekeeping sortase produced by the important human pathogen Streptococcus pyogenes, the crystal structure of this protein has been solved and its transpeptidase activity established in vitro. The structure reveals a novel arrangement of key catalytic residues in the active site of a sortase, the first that is consistent with kinetic analysis. The structure also provides a complete description of residue positions surrounding the active site, overcoming the limitation of localized disorder in previous structures of sortase A-type proteins. Modification of the active site Cys through oxidation to its sulfenic acid form or by an alkylating reagent supports a role for a reactive thiol/thiolate in the catalytic mechanism. These new insights into sortase structure and function could have important consequences for inhibitor design.
Assuntos
Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Streptococcus pyogenes/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Cinética , Oxirredução , Estrutura Terciária de Proteína , Ácidos Sulfênicos/químicaRESUMO
Bacillus subtilis StoA is an extracytoplasmic thiol-disulfide oxidoreductase (TDOR) important for the synthesis of the endospore peptidoglycan cortex protective layer. Here we demonstrate that StoA is membrane-associated in B. subtilis and report the crystal structure of the soluble protein lacking its membrane anchor. This showed that StoA adopts a thioredoxin-like fold with N-terminal and internal additions that are characteristic of extracytoplasmic TDORs. The CXXC active site of the crystallized protein was found to be in a mixture of oxidized and reduced states, illustrating that there is little conformational variation between redox states. The midpoint reduction potential was determined as -248 mV versus normal hydrogen electrode at pH 7 consistent with StoA fulfilling a reductive role in endospore biogenesis. pK(a) values of the active site cysteines, Cys-65 and Cys-68, were determined to be 5.5 and 7.8. Although Cys-68 is buried within the structure, both cysteines were found to be accessible to cysteine-specific alkylating reagents. In vivo studies of site-directed variants of StoA revealed that the active site cysteines are functionally important, as is Glu-71, which lies close to the active site and is conserved in many reducing extracytoplasmic TDORs. The structure and biophysical properties of StoA are very similar to those of ResA, a B. subtilis extracytoplasmic TDOR involved in cytochrome c maturation, raising important general questions about how these similar but non-redundant proteins achieve specificity. A detailed comparison of the two proteins demonstrates that relatively subtle differences, largely located around the active sites of the proteins, are sufficient to confer specificity.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Membrana/química , Esporos Bacterianos/química , Sequência de Aminoácidos , Biofísica/métodos , Domínio Catalítico , Citocromos c/química , Ácido Glutâmico/química , Concentração de Íons de Hidrogênio , Proteínas de Membrana/fisiologia , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, DeltapK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.
Assuntos
Substituição de Aminoácidos/genética , Bacillus subtilis , Líquido Extracelular/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Motivos de Aminoácidos/genética , Substituição de Aminoácidos/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Líquido Extracelular/química , Concentração de Íons de Hidrogênio , Oxirredução , Proteína Dissulfeto Redutase (Glutationa)/química , Proteína Dissulfeto Redutase (Glutationa)/genética , Proteína Dissulfeto Redutase (Glutationa)/metabolismoRESUMO
ResA, an extracytoplasmic thioredoxin from Bacillus subtilis, acts in cytochrome c maturation by reducing the disulfide bond present in apocytochromes prior to covalent attachment of heme. This reaction is (and has to be) specific, as broad substrate specificity would result in unproductive shortcircuiting with the general oxidizing thioredoxin(s) present in the same compartment. Using mutational analysis and subsequent biochemical and structural characterization of active site variants, we show that reduced ResA displays unusually low reactivity at neutral pH, consistent with the observed high pKa values>8 for both active site cysteines. Residue Glu80 is shown to play a key role in controlling the acid-base properties of the active site. A model in which substrate binding dramatically enhances the reactivity of the active site cysteines is proposed to account for the specificity of the protein. Such a substratemediated activation mechanism is likely to have wide relevance for extracytoplasmic thioredoxins.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoRESUMO
Post-translational maturation of cytochromes c involves the covalent attachment of heme to the Cys-Xxx-Xxx-Cys-His motif of the apo-cytochrome. For this process, the two cysteines of the motif must be in the reduced state. In bacteria, this is achieved by dedicated, membrane-bound thiol-disulfide oxidoreductases with a high reducing power, which are essential components of cytochrome c maturation systems and are also linked to cellular disulfide-bond formation machineries. Here we report high-resolution structures of oxidized and reduced states of a soluble, functional domain of one such oxidoreductase, ResA, from Bacillus subtilis. The structures elucidate the structural basis of the protein's high reducing power and reveal the largest redox-coupled conformational changes observed to date in any thioredoxin-like protein. These redox-coupled changes alter the protein surface and illustrate how the redox state of ResA predetermines to which substrate it binds. Furthermore, a polar cavity, present only in the reduced state, may confer specificity to recognize apo-cytochrome c. The described features of ResA are likely to be general for bacterial cytochrome c maturation systems.