Native Isolation of the CcsB Protein from Synechocystis sp. PCC 6803 Involved in Cytochrome f Maturation in Cyanobacteria and Plastids

The last step for biosynthesis of c type cytochromes, indispensable for photosynthesis in cyanobacteria and plants, involves heme transport across the membrane and its covalent attachment to the apoprotein. In cyanobacteria, heme attachment occurs in the thylakoid lumen and probably also in the periplasm and requires at least four proteins, believed to be organized in intrinsic membrane protein complex. To allow isolation and identification of such complex, CcsB protein was tagged with 6xHis tag on its N terminus and expressed under the strong psbAII promoter in the cyanobacterium Synechocystis sp. PCC 6803. Similarly, CcsA protein was tagged with FLAG tag under the control of the same promoter. Although expression of both proteins under strong cyanobacterial promoter did not increase steady state contents of the CcsB protein, the fusion tags did not influence properties of the CcsB and CcsA proteins and the resulting mutants had the same phenotype as the wild type. Protein fraction containing CcsBHis protein was partially isolated from the solubilised membranes under native conditions.     

The reactivity of cytochrome c with soft ligands

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron—methionine sulfur bond of this heme protein is enhanced by delocalization of the metal l₂, electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.     

Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c

Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80–heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80–heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c.     

Maturation of a eukaryotic cytochrome <Emphasis Type="Italic">c</Emphasis> in the cytoplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis> without the assistance by a dedicated biogenesis apparatus

Maturation of c-type cytochromes involves the covalent and stereospecific enzymatic attachment of a heme b via thioether linkages to two conserved cysteines within apocytochromes. Horse cytochrome c is readily matured into its native holoform in the cytoplasm of E. coli when co-expressed with yeast cytochrome c heme lyase. Here we report the low yield formation of holocytochrome with covalently attached heme also in the absence of heme lyase. This is the first demonstration of in vivo maturation of a eukaryotic cytochrome c in a prokaryotic cytoplasm without the assistance by a dedicated enzymatic maturation system. The assembled cytochrome c can be oxidized by cytochrome c oxidase, indicating the formation of a functional protein. The absorption spectrum is typical of a low spin, six coordinated c-type heme. Nevertheless, minor spectral differences relative to the native cytochrome c, deviation of the midpoint reduction potential and slightly altered kinetic parameters of the interaction with cytochrome c oxidase emphasize the importance of cytochrome c heme lyase in folding cytochrome c into its native conformation.     

Overexpression, characterization, and crystallization of the functional domain of cytochrome cz from Chlorobium tepidum

Cytochrome c _z is found in green sulfur photosynthetic bacteria, and is considered to be the only electron donor to the special pair P840 of the reaction center. It consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. Large scale expression of the C-terminal functional domain of the cytochrome c _z (C-cyt c _z) from the thermophilic bacterium Chlorobium tepidum has been achieved using the Escherichia coli expression system. The C-cyt c _z expressed has been highly purified, and is stable at room temperature over 10 days of incubation for both reduced and oxidized forms. Spectroscopic measurements indicate that the heme iron in C-cyt c _z is in a low-spin state and this does not change with the redox state. ¹H-NMR spectra of the oxidized C-cyt c _z exhibited unusually large paramagnetic chemical shifts for the heme methyl protons in comparison with those of other Class I ferric cytochromes c. Differences in the ¹H-NMR linewidth were observed for some resonances, indicating different dynamic environments for these protons. Crystals of the oxidized C-cyt c _z were obtained using ammonium sulfate as a precipitant. The crystals diffracted X-rays to a maximum resolution of 1.2 ?, and the diffraction data were collected to 1.3 ? resolution.     

The roles of different regions of the CycH protein in<Emphasis Type="Italic"> c</Emphasis>-type cytochrome biogenesis in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Cytochrome c heme lyases encoded by the Sinorhizobium meliloti cycHJKL operon are responsible for generating the covalent bond between the heme prosthetic group and apocytochromes c. The CycH protein with its presumably membrane-associated N-terminal and periplasmic C-terminal parts is thought to be responsible for binding apocytochrome and presenting it to the heme ligation machinery. We propose that these two modules of CycH play roles in different functions of the protein. The N-terminal 96 amino acids represent an active subdomain of the protein, which is able to complement the protoporphyrin IX (PPIX) accumulation phenotype of the cycH mutant strain AT342, suggesting that it is involved in the final steps of heme C biosynthesis. Furthermore, three tetratricopeptide (TPR) domains have been identified in the C-terminal periplasmic region of the CycH protein. TPR domains are known to mediate protein-protein interactions. Each of these CycH domains is absolutely required for protein function, since plasmid constructs carrying cycH genes with in-frame TPR deletions were not able to complement cycH mutants for their nitrate reductase (Rnr^–) and nitrogen-fixing (Fix^–) phenotypes. We also found that the 309-amino acid N-terminal portion of the CycH, which includes all the TPR domains, is able to mediate the assembly of the c-type cytochromes required for the Rnr⁺ phenotype. In contrast, only the full-length protein confers the ability to fix nitrogen.Communicated by A. Kondorosi     

Crystal structure and biochemical features of EfeB/YcdB from Escherichia coli O157: ASP235 plays divergent roles in different enzyme-catalyzed processes

EfeB/YcdB is a member of the dye-decolorizing peroxidase (DyP) protein family. A recent study has shown that this protein can extract iron from heme without breaking the tetrapyrrole ring. We report the crystal structure of EfeB from Escherichia coli O157 bound to heme at 1.95 Å resolution. The EfeB monomer contains two domains. The heme molecule is located in a large hydrophobic pocket in the C-terminal domain. A long loop connecting the two domains extensively interacts with the heme, which is a distinctive structural feature of EfeB homologues. A large tunnel formed by this loop and the β-sheet of C-terminal domain provides a potential cofactor/substrate binding site. Biochemical data show that the production of protoporphyrin IX (PPIX) is closely related to the peroxidation activity. The mutant D235N keeps nearly the same activity of guaiacol peroxidase as the wild-type protein, whereas the corresponding mutation in the classic DyP protein family completely abolished the peroxidation activity. These results suggest that EfeB is a unique member of the DyP protein family. In addition, dramatically enhanced fluorescence excitation and emission of EfeB-PPIX was observed, implying this protein may be used as a red color fluorescence marker.     

The biosynthesis of bacterial and plastidic c-type cytochromes

The biosynthesis of bacterial and plastidic c-type cytochromes includes several steps that occur post-translationally. In the case of bacterial cytochromes, the cytosolically synthesized pre-proteins are translocated across the cytoplasmic membrane, the pre-proteins are cleaved to their mature forms and heme is ligated to the processed apoprotein. Although heme attachment has not been studied extensively at the biochemical level, molecular genetic approaches suggest that the reaction generally occurs after translocation of the apoprotein to the periplasm. Recent studies with Bradyrhizobium japonicum and Rhodobacter capsulatus indicate that the process of heme attachment requires the function of a large number of genes. Mutation of these genes generates a pleiotropic deficiency in all c-type cytochromes, suggesting that the gene products participate in processes required for the biosynthesis of all c-type cytochromes. In eukaryotic cells, the biosynthesis of photosynthetic c-type cytochromes is somewhat more complex owing to the additional level of compartmentation. Nevertheless, the basic features of the pathway appear to be conserved. For instance, as is the case in bacteria, translocation and processing of the pre-proteins is not dependent on heme attachment. Genetic analysis suggests that the nuclear as well as the plastid genomes encode functions required for heme attachment, and that these genes function in the biosynthesis of the membrane-associated as well as the soluble c-type cytochrome of chloroplasts. A feature of cytochromes c biogenesis that appears to be conserved between chloroplasts and mitochondria is the sub-cellular location of the heme attachment reaction (p-side of the energy transducing membrane). Continued investigation of all three experimental systems (bacteria, chloroplasts, mitochondria) is likely to lead to a greater understanding of the biochemistry of cytochrome maturation as well as the more general problem of cofactor-protein association during the assembly of an energy transducing membrane.Abbreviations CCHL cytochrome c/heme lyase - CC₁HL cytochrome cl/heme lyase - cyt cytochrome - EMS ethyl methane sulphonate - n-side electrochemically negative side of an energy transducing membrane - p-side electrochemically positive side of an energy transducing membrane - PhoA alkaline phosphatase (encoded by the phoA locus)     

A heme fusion tag for protein affinity purification and quantification

We report a novel affinity‐based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L ‐histidine‐immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme‐tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag‐HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.     

Cloning,expression, and physicochemical characterization of a new diheme cytochrome <Emphasis Type="Italic">c</Emphasis> from <Emphasis Type="Italic">Shewanella baltica</Emphasis> OS155

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV–vis, magnetic circular dichroism, and ¹H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid–base equilibria with pK _a values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of −0.144 and −0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye–Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°′.     

Characterization of cytochrome oxidase purified from rat liver

The purpose of this study was to characterize the physical properties of cytochromec oxidase from rat liver. The enzyme was extracted from isolated mitochondria with nonionic detergents and further purified by ion-exchange chromatography on DEAE Bio-Gel A. The purified enzyme contained 9.64 nmol heme a/mg protein and one iron atom plus one copper atom for each heme a. The specific activity of the final preparation was 146 µmol of ferrocytochromec oxidized/min · mg protein, measured at pH 5.7. The spectral properties of the enzyme were characteristic of purified cytochrome oxidase and indicated that the preparation was free of cytochromesb, c, andc ₁. In analytical ultracentrifugation studies, the enzyme sedimented as a single component with anS ^20,w of5.35S. The Stokes radius of the enzyme was determined by gel filtration chromatography and was equal to 75 Å. The molecular weight of the oxidase calculated from its sedimentation coefficient and Stokes' radius was 180,000, indicating that the active enzyme contained two heme a groups. The purified cytochrome oxidase was also subjected to dodecyl sulfate-polyacrylamide gel electrophoresis in order to determine its components. The enzyme was resolved into five polypeptides with the molecular weights of I, 27,100; II, 15,000; III, 11,900; IV 9800; and V, 9000.     

Crystallographic structure of Rhodospirillum molischianum ferricytochrome c' at 2.5 A resolution

This paper describes the 2.5 Å crystallographic structure determination of ferricytochrome c′ from the photosynthetic bacterium Rhodospirillum molischianum. The molecule is a symmetric dimer, with each 128-residue polypeptide chain incorporating a covalently bound protoheme IX prosthetic group. The monomer is structurally organized as an array of four nearly parallel α-helices, which pack most closely at one end and thereafter spatially diverge to accommodate the heme prosthetic group. Although local features of the heme attachment pattern resemble those seen in cytochrome c, the heme iron in cytochrome c′ is pentaco-ordinate with a solvent-exposed histidine residue furnishing the single axial ligand to the heme iron.Subunit association in the dimeric molecule is principally stabilized by helix interactions, which are qualitatively similar to those occurring within each monomer. These interactions result in a dimer geometry that situates the exposed regions of both hemes on the same molecular surface.The structural basis for some of the physiochemical properties cytochrome c′ are examined and compared to those of other heme proteins of known structure.     

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539–552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10²–10³ M⁻¹ s⁻¹ and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.     

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli��Expression and characterization of cytochrome-tagged Complex I subunits

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane‐spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C‐terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c₅₅₀. Compared with other available fusion‐protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter‐like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo‐cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c₅₅₀ domain in all the fusion proteins exhibited normal spectra and redox properties, with an E_m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c‐tag. Finally, a his‐tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.     

Ascorbate peroxidase activity of cytochrome c

Abstract

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10–60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.     

Heme ladder, a direct molecular weight marker for immunoblot analysis

Detection methods for immunoblot analysis are often based on peroxidase conjugates. However, molecular weight markers directly detectable for general use in such systems are not available. Here, we describe the preparation of a direct molecular weight marker consisting of heme-tagged proteins, whose enzymatic activities make them detectable simultaneously with the antigen in peroxidase-based immunoblot systems. The peroxidase activity results from the covalent attachment of heme to selected engineered periplasmic proteins, catalyzed by the cytochrome c maturation system of Escherichia coli. The newly designed heme-tagged proteins were combined with a previously constructed heme-tagged maltose-binding protein and cytochrome c. The resulting heme ladder was shown to be suitable as a protein standard for direct molecular weight estimation in immunoblot analysis due to the peroxidase activity of its constituents. The heme ladder consists of proteins between 12 and 85 kDa and can be produced at low cost. The marker was stable when kept at 4, −20, and −80 °C for >6 months.     

Cytochrome c Biogenesis: Mechanisms for Covalent Modifications and Trafficking of Heme and for Heme-Iron Redox Control

Summary: Heme is the prosthetic group for cytochromes, which are directly involved in oxidation/reduction reactions inside and outside the cell. Many cytochromes contain heme with covalent additions at one or both vinyl groups. These include farnesylation at one vinyl in hemes o and a and thioether linkages to each vinyl in cytochrome c (at CXXCH of the protein). Here we review the mechanisms for these covalent attachments, with emphasis on the three unique cytochrome c assembly pathways called systems I, II, and III. All proteins in system I (called Ccm proteins) and system II (Ccs proteins) are integral membrane proteins. Recent biochemical analyses suggest mechanisms for heme channeling to the outside, heme-iron redox control, and attachment to the CXXCH. For system II, the CcsB and CcsA proteins form a cytochrome c synthetase complex which specifically channels heme to an external heme binding domain; in this conserved tryptophan-rich “WWD domain” (in CcsA), the heme is maintained in the reduced state by two external histidines and then ligated to the CXXCH motif. In system I, a two-step process is described. Step 1 is the CcmABCD-mediated synthesis and release of oxidized holoCcmE (heme in the Fe⁺³ state). We describe how external histidines in CcmC are involved in heme attachment to CcmE, and the chemical mechanism to form oxidized holoCcmE is discussed. Step 2 includes the CcmFH-mediated reduction (to Fe⁺²) of holoCcmE and ligation of the heme to CXXCH. The evolutionary and ecological advantages for each system are discussed with respect to iron limitation and oxidizing environments.     

纯化标签的不同位置对Δ6脂肪酸脱饱和酶异源表达的影响

【背景】目前利用酵母表达系统已鉴定了多种物种中的Δ6脂肪酸脱饱和酶(FADS6)。由于FADS6是一种具有多个跨膜螺旋的膜蛋白,使得其大量表达和纯化具有挑战性。【目的】探索FADS6的高效表达策略,研究纯化标签添加的位置对高山被孢霉FADS6I (Ma FADS6I)重组表达效率的影响。【方法】在毕赤酵母表达载体中插入串联亲和标签HRV 3C-Protein A-His,利用改造后的载体构建带有N端或C端标签的Ma FADS6I表达载体;通过电转化获得毕赤酵母重组表达菌株;利用斑点印迹杂交(DotBlot)、聚丙烯酰胺凝胶电泳(SDS-PolyacrylamideGelElectrophoresis,SDS-PAGE)和免疫印迹(Western Blot)分析重组蛋白的表达水平,并利用气相色谱-质谱(Gas Chromatography-Mass Spectrometry,GC-MS)分析检测Ma FADS6I催化生成的脂肪酸。【结果】通过大量的毕赤酵母转化子筛选,最终获得高效表达Ma FADS6I的毕赤酵母重组菌,证实各转化子的表达具有差异性,Ma FADS6I的C端带有纯化标签较N端更有利于表达。【结论】在Ma FADS6I的C端添加纯化标签比在N端添加更有利于该蛋白在酵母系统中的表达以及底物的转化,为进一步探究FADS6高效表达和结构功能奠定了基础。     

Tailoring of recombinant FDH: effect of histidine tag location on solubility and catalytic properties of Chaetomium thermophilum formate dehydrogenase (CtFDH)

Abstract

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD⁺ dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD⁺ to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.     

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.