Effects of starvation for heme on the synthesis of porphyrins in Escherichia coli

A study is described of the regulation of porphyrin synthesis in Escherichia coli using a heme-permeable, hemH deletion mutant, designated VS212. This strain utilizes only exogenous hemin that is supplied in the medium and accumulates porphyrins since the final step in the synthesis of heme is genetically blocked. It is possible, therefore, to monitor the rate of synthesis of heme by examining the accumulation of porphyrins. Using this system, we found that the rate of production of porphyrins depended on the availability of heme. The lower the concentration of hemin in the medium, the higher the level of porphyrins that accumulated. We next examined the mechanism responsible for the activation of porphyrin synthesis upon starvation for heme. The main activation occurred at the step that leads to the synthesis of 5-aminolevulinic acid (ALA). Starvation for heme induced the expression of a hemA-lacZ fusion gene, as previously reported, but an activation pathway that is independent of the hemA promoter was also identified. We found that starvation for heme caused the stringent response, and such starvation promoted the synthesis of porphyrins without having any effect on the expression of the hemA-lacZ fusion gene. We suggest a model for the regulation of porphyrin synthesis whereby the synthesis of porphyrins is coordinated with that of proteins. Received: 28 January 1997 / Accepted: 13 March 1997     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Sn-protoporphyrin inhibits both heme degradation and hemozoin formation in Rhodnius prolixus midgut

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.     

Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa , which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M_r = 37 008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5′ end of the hemB mRNA was determined and the −10 and −35 regions of a potential σ⁷⁰-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg²⁺-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg²⁺-dependent activity was directly demonstrated. Received: 11 July 1997 / Accepted: 9 October 1997     

Isolation of Hem3 mutants from Candida albicans by sequential gene disruption

Summary Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a 2.0 kb fragment by subcloning and a BglII site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the BglII site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura⁺, Leu⁺ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.     

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e⁻_aq) and carboxyl anion radical (CO₂) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e⁻_aq with the negatively charged ferricytochrome c-551 (17 nM⁻¹ · s⁻¹) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM⁻ · s⁻). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e⁻_aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 10⁴ s⁻¹) observed after fast e⁻_aq reduction is attributed to a small conformational change. The rate of reaction of CO₂ with ferricytochrome c-551 (0.7 nM⁻¹ · s⁻) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.     

Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the anaerobic bacterium Desulfovibrio desulfuricans

A bacterioferritin was recently isolated from the anaerobic sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 [Romão et al. (2000) Biochemistry 39, 6841–6849]. Although its properties are in general similar to those of the other bacterioferritins, it contains a haem quite distinct from the haem B, found in bacterioferritins from aerobic organisms. Using visible and NMR spectroscopies, as well as mass spectrometry analysis, the haem is now unambiguously identified as iron-coproporphyrin III, the first example of such a prosthetic group in a biological system. This unexpected finding is discussed in the framework of haem biosynthetic pathways in anaerobes and particularly in sulphate-reducing bacteria.     

The Rhizobium etli FixL protein differs in structure from other known FixL proteins

The central heme-binding domain in the FixL proteins of Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum biovar viciae and Azorhizobium caulinodans, is highly conserved. The similarity with the corresponding domain in the Rhizobium etli FixL protein is considerably less. This observation prompted us to analyze the heme-binding capacities of the R. etli FixL protein. The R. etlifixL gene was overexpressed in Escherichia coli. In the presence of S. meliloti FixJ, the overexpressed R. etli FixL protein was able to enhance FixJ-mediated activation of an S. meliloti pnifA-lacZ fusion, indicating that the R.␣etli FixL protein possesses an active conformation in E. coli. Subsequently, using a non-denaturing gel assay for heme, we analyzed the heme-binding capacity of the R.␣etli FixL protein expressed in E. coli, taking the S.␣meliloti FixL protein as a positive control. The R. etli FixL protein expressed in E. coli does not contain a heme group, in contrast to the S. meliloti FixL protein. Therefore we conclude that the R. etli FixL is a non-heme protein in the nif regulatory cascade. Received: 22 August 1997 / Accepted: 20 October 1997     

Genetic control of chlorophyll biosynthesis: Regulation of delta-aminolevulinate synthesis in Chlamydomonas

Summary A chlorophyll-deficient mutant, br _s -1, of Chlamydomonas reinhardtii has been shown to accumulate low levels of an intermediate, protoporphyrin (PROTO), and to form light-brown colonies. A double mutant, br _s -1 r-1, accumulates 15-fold more PROTO than br _s -1 and forms dark-brown colonies. Enzymes synthesizing the first intermediate of chlorophyll, delta-aminolevulinate (ALA), from these two mutants and the wild-type are equally sensitive to inhibition by heme. The activity of ALA-synthesizing enzymes from br _s -1 r-1 is similar to that of the wild-type and is more than threefold that of br _s -1. It is proposed that the ALA-synthesizing enzymes in br _s -1 are under repression while r-1 is a mutation of the regulatory gene and consequently derepresses the synthesis of its own ALA-synthesizing enzymes. In addition, by mutagenizing br _s -1, we isolated six more double mutants having the same phenotype as br _s -1 r-1. Five of them are identical to br _s -1 r-1, the remaining one (db-10) carries a second mutation nonallelic to r-1. The ALA-synthesizing enzymes from db-10 are much less sensitive to heme inhibition than those from the wild type. It is proposed that ALA synthesis in Clamydomonas is regulated both allosterically and genetically.Abbreviations PROTO protoporphyrin - ALA delta-aminolevulinate - Mg-PROTO magnesium-protoporphyrin - GSA glutamate-1-semialdehyde     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 > Lys 25 Lys 79 ≈ Lys 87 > Lys 8 ≈ Lys 27 ≈ Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.     

Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis

The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.     

Molecular dynamics in cytochrome c oxidase Mössbauer spectra deconvolution

In this work low temperature molecular dynamics simulations of cytochrome c oxidase are used to predict an experimentally observable, namely Mössbauer spectra width. Predicted lineshapes are used to model Lorentzian doublets, with which published cytochrome c oxidase Mössbauer spectra were simulated. Molecular dynamics imposed constraints to spectral lineshapes permit to obtain useful information, like the presence of multiple chemical species in the binuclear center of cytochrome c oxidase. Moreover, a benchmark of quality for molecular dynamic simulations can be obtained. Despite the overwhelming importance of dynamics in electron–proton transfer systems, limited work has been devoted to unravel how much realistic are molecular dynamics simulations results. In this work, molecular dynamics based predictions are found to be in good agreement with published experimental spectra, showing that we can confidently rely on actual simulations. Molecular dynamics based deconvolution of Mössbauer spectra will lead to a renewed interest for application of this approach in bioenergetics.     

A simple method for the determination of reduction potentials in heme proteins

We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (E_m = −115 mV ± 2 mV, pH 7.0) and human CLOCK (E_m = −111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.     

Temperature dependence in the absorption spectra of beef liver catalase

The spin characteristics of the ferric heme groups in native beef liver catalase. and in the complexes formed by reaction with fluoride, cyanide, azide, thiocyanate, and cyanate ions have been studied using absorption spectroscopy over the temperature range of 4–285 K. The azide. isothiocyanate, and isocyanate complexes of catalase are considered to be high-spin ferric heme complexes at room temperature, but undergo a thermal spin change below 300 K. The temperature dependence of these absorption spectra, however, cannot be analyzed in terms of simple Boltzmann distributions between two S = $\text{1}\text{2}$ and S = $\text{5}\text{2}$ spin stales. The data show that these spin changes occur over a very narrow temperature range, but do not result in the formation of completely, low-spin complexes. The data also suggest that the thermal spin changes that occur below the glassing temperature of the solvent are dependent upon the conformational changes which take place within the protein itself with a change in temperature, and which directly affect the environment of the heme group.     

Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b₅₉₅, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b₅₉₅ and heme b₅₅₈. It is concluded that 1) O₂ binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O₂ binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b₅₉₅, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm]?→?E107?→?E99 (heme b₅₉₅)?→?E445 (heme d)?→?oxygenated heme d.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.     

Multiple invasions of Gypsy and Micropia retroelements in genus Zaprionus and melanogaster subgroup of the genus Drosophila

Background  

The Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus.