Lars Hederstedt

Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis

BACKGROUND The metallation of closed ring tetrapyrroles resulting in the formation of hemes, chlorophylls and vitamin B12 is catalyzed by specific enzymes called chelatases. Ferrochelatase catalyzes the terminal step in heme biosynthesis by inserting ferrous ion into protoporphyrin IX by a mechanism that is poorly understood. Mutations in the human gene for ferrochelatase can result in the disease erythropoietic protoporphyria, and a further understanding of the mechanism of this enzyme is therefore of clinical interest. No three-dimensional structure of a tetrapyrrole metallation enzyme has been available until now. RESULTS The three-dimensional structure of Bacillus subtilis ferrochelatase has been determined at 1.9 A resolution by the method of multiple isomorphous replacement. The structural model contains 308 of the 310 amino acid residues of the protein and 198 solvent molecules. The polypeptide is folded into two similar domains each with a four-stranded parallel beta sheet flanked by alpha helices. Structural elements from both domains build up a cleft, which contains several amino acid residues that are invariant in ferrochelatases from different organisms. In crystals soaked with gold and cadmium salt solutions, the metal ion was found to be coordinated to the conserved residue His 183, which is located in the cleft. This histidine residue has previously been suggested to be involved in ferrous ion binding. CONCLUSIONS Ferrochelatase seems to have a structurally conserved core region that is common to the enzyme from bacteria, plants and mammals. We propose that porphyrin binds in the identified cleft; this cleft also includes the metal-binding site of the enzyme. It is likely that the structure of the cleft region will have different conformations upon substrate binding and release.

A structural moDAl for the membrane-integral domain of succinate:quinone oxidoreductases

Many succinate:quinone oxidoreductases in bacteria and mitochondria, i.e. succinate:quinone reductases and fumarate reductases, contain in the membrane anchor a cytochrome b whose structure and function is poorly understood. Based on biochemical data and polypeptiDA sequence information, we show that the anchors in different organisms are related DAspite an apparent diversity in polypeptiDA and heme composition. A general structural moDAl for the membrane‐integral domain of the anchors is proposed. It is an antiparallel four‐helix bundle with a novel arrangement of hexa‐coordinated protoheme IX. The structure can be applied to a larger group of membrane‐integral cytochromes of b‐type and has evolutionary and functional implications.

The physical state of water in bacterial spores

The bacterial spore, the hardiest known life form, can survive in a metabolically dormant state for many years and can withstand high temperatures, radiation, and toxic chemicals. The molecular basis of spore dormancy and resistance is not understood, but the physical state of water in the different spore compartments is thought to play a key role. To characterize this water in situ, we recorded the water 2H and 17O spin relaxation rates in D2O-exchanged Bacillus subtilis spores over a wide frequency range. The data indicate high water mobility throughout the spore, comparable with binary protein–water systems at similar hydration levels. Even in the dense core, the average water rotational correlation time is only 50 ps. Spore dormancy therefore cannot be explained by glass-like quenching of molecular diffusion but may be linked to dehydration-induced conformational changes in key enzymes. The data demonstrate that most spore proteins are rotationally immobilized, which may contribute to heat resistance by preventing heat-denatured proteins from aggregating irreversibly. We also find that the water permeability of the inner membrane is at least 2 orders of magnitude lower than for model membranes, consistent with the reported high degree of lipid immobilization in this membrane and with its proposed role in spore resistance to chemicals that damage DNA. The quantitative results reported here on water mobility and transport provide important clues about the mechanism of spore dormancy and resistance, with relevance to food preservation, disease prevention, and astrobiology.

Genes required for cytochrome c synthesis in Bacillus subtilis

Cytochromes of c‐type contain covalently bound haem and in bacteria are located on the periplasmic side of the cytoplasmic membrane. More than eight different gene products have been identified as being specifically required for the synthesis of cytochromes c in Gram‐negative bacteria. Corresponding genes are not found in the genome sequences of Gram‐positive bacteria. Using two random mutagenesis approaches, we have searched for cytochrome c biogenesis genes in the Gram‐positive bacterium Bacillus subtilis. Three genes, resB, resC and ccdA, were identified. CcdA has been found previously and is required for a late step in cytochrome c synthesis and also plays a role in spore synthesis. No function has previously been assigned for ResB and ResC but these predicted membrane proteins show sequence similarity to proteins required for cytochrome c synthesis in chloroplasts. Attempts to inactivate resB and resC in B. subtilis have indicated that these genes are essential for growth. We demonstrate that various nonsense mutations in resB or resC can block synthesis of cytochromes c with no effect on other types of cytochromes and little effect on sporulation and growth. The results strongly support the recent proposal that Gram‐positive bacteria, cyanobacteria, ε‐proteobacteria, and chloroplasts have a similar type of machinery for cytochrome c synthesis (System II), which is very different from those of most Gram‐negative bacteria (System I) and mitochondria (System III).

Mutations in the Thiol-Disulfide Oxidoreductases BdbC and BdbD Can Suppress Cytochrome c Deficiency of CcdA-Defective Bacillus subtilis Cells

Cytochromes of the c type in the gram-positive bacterium Bacillus subtilis are all membrane anchored, with their heme domains exposed on the outer side of the cytoplasmic membrane. They are distinguished from other cytochromes by having heme covalently attached by two thioether bonds. The cysteinyls in the heme-binding site (CXXCH) in apocytochrome c must be reduced in order for the covalent attachment of the heme to occur. It has been proposed that CcdA, a membrane protein, transfers reducing equivalents from thioredoxin in the cytoplasm to proteins on the outer side of the cytoplasmic membrane. Strains deficient in the CcdA protein are defective in cytochrome c and spore synthesis. We have discovered that mutations in the bdbC and bdbD genes can suppress the defects caused by lack of CcdA. BdbC and BdbD are thiol-disulfide oxidoreductases. Our experimental findings indicate that these B. subtilis proteins functionally correspond to the well-characterized Escherichia coli DsbB and DsbA proteins, which catalyze the formation of disulfide bonds in proteins in the periplasmic space.

Bacillus subtilis CtaA and CtaB function in haem A biosynthesis

Haem A, a prosthetic group of many respiratory oxidases, is probably synthesized from haem B (protohaem IX) in a pathway in which haem O is an intermediate. Possible roles of the Bacillus subtilis ctaA and CtaB gene products in haem O and haem A synthesis were studied. Escherichia coli does not contain haem A. The CtaA gene on plasmids in E. coli resulted in haem A accumulation in membranes. The presence of CtaB together with ctaA increased the amount of haem A found in E. coli. Haem O was not detected in wild‐type B. subtilis strains. A previously isolated B. subtilis CtaA deletion mutant was found to contain haem B and haem O, but not haem A. B. subtilis ctaB deletion mutants were constructed and found to tack both haem A and haem O. The results with E. coli and B. subtilis strongly suggest that the B. subtilis CtaA protein functions in haem A synthesis. It is tentatively suggested that it functions in the oxygeNatlon/oxidation of the methyl side group of carbon 8 of haem O. B. subtilis CtaB, which is homologous to Saccharomyces cerevisiae COX10 and E. coli CyoE, also has a role in haem A synthesis and seems to be required for both cytochrome a and cytochrome o synthesis.

Chapter 7 Progress in succinate:quinone oxidoreductase research

Publisher Summary This chapter discusses the progress in succinate:quinone oxidoreductase research. It reviews the progress made mainly within the last decade in understanding of the genetics, biogenesis, structure and functions of succinate:quinone oxidoreductases. The work on this class of enzymes has involved a vast amount of experimental efforts in many laboratories. As in many other fields of biological research, rapid advances have resulted from the increased use of a molecular biologists approach, that is, a combination of molecular genetics, biochemistry and biophysical techniques. Succinate:quinone oxidoreductases are membrane bound enzymes that can catalyze the oxidation of succinate to fumarate coupled to the reduction of a quinone and the reduction of fumarate to succinate coupled to the oxidation of quinol. Succinate:quinone reductase (SQR), is present in strictly aerobic cells, and in vivo predominantly catalyzes the oxidation of succinate. Continued investigations of the structure of SQR and QFR enzymes will provide detailed, three-dimensional structural information, which is required for the better understanding of mechanisms of catalysis at the dicarboxylate and the quinone active sites and in the intra-molecular electron transfer.

Enterococcus faecalis Heme-Dependent Catalase

Enterococcus faecalis cells cannot synthesize porphyrins and do not rely on heme for growth but can take up heme and use it to synthesize heme proteins. We recently described a cytochrome bd in E. faecalis strain V583 and here report the identification of a chromosomal gene, katA, encoding a heme-containing cytoplasmic catalase. The 54-kDa KatA polypeptide shows sequence similarity to members of the family of monofunctional catalases. A hexahistidyl-tagged version of the catalase was purified, and major characteristics of the enzyme were determined. It contains one protoheme IX group per KatA polypeptide. Catalase activity was detected only in E. faecalis cells grown in the presence of heme in the medium; about 2 and 10 micro M hemin was required for half-maximal and maximal production of catalase, respectively. Our finding of a catalase whose synthesis is dependent on the acquisition of heme in the opportunistic pathogen E. faecalis might be of clinical importance. Studies of cellular heme transport and heme protein assembly and in vivo synthesis of metalloprotein analogs for biotechnological applications are impeded by the lack of experimental systems. We conclude that the E. faecalis cell potentially provides such a desired system.

Organization of genes for tetrapyrrole biosynthesis in Gram-positive bacteria

Clusters of genes encoding enzymes for tetrapyrrole biosynthesis were cloned from Bacillus sphaericus, Bacillus stearothermophilus, Brevibacillus brevis and Paenibacillus macerans. The sequences of all hemX genes found, and of a 6.3 kbp hem gene cluster from P. macerans, were determined. The structure of the hem gene clusters was compared to that of other Gram-positive bacteria. The Bacillus and Brevibacillus species have a conserved organization of the genes hemAXCDBL, required for biosynthesis of uroporphyrinogen III (UroIII) from glutamyl-tRNA. In P. macerans, the hem genes for UroIII synthesis are also closely linked but their organization is different: there is no hemX gene and the gene cluster also contains genes, cysG8 and cysG(A)-hemD, encoding the enzymes required for synthesis of sirohaem from UroIII. Bacillus subtilis contains genes for three proteins, NasF, YInD and YInF, with sequence similarity to Escherichia coli CysG, which is a multi-functional protein catalysing sirohaem synthesis from UroIII. It is shown that YInF is required for sirohaem synthesis and probably catalyses the precorrin-2 to sirohaem conversion. YInD probably catalyses precorrin-2 synthesis from UroIII and NasF seems to be specific for nitrite reduction.

Composition and function of cytochrome c biogenesis System II.

Organisms employ one of several different enzyme systems to mature cytochromes c. The biosynthetic process involves the periplasmic reduction of cysteine residues in the heme c attachment motif of the apocytochrome, transmembrane transport of heme b and stereospecific covalent heme attachment via thioether bonds. The biogenesis System II (or Ccs system) is employed by β‐, δ‐ and ε‐proteobacteria, Gram‐positive bacteria, Aquificales and cyanobacteria, as well as by algal and plant chloroplasts. System II comprises four (sometimes only three) membrane‐bound proteins: CcsA (or ResC) and CcsB (ResB) are the components of the cytochrome c synthase, whereas CcdA and CcsX (ResA) function in the generation of a reduced heme c attachment motif. Some ε‐proteobacteria contain CcsBA fusion proteins constituting single polypeptide cytochrome c synthases especially amenable for functional studies. This minireview highlights the recent findings on the structure, function and specificity of individual System II components and outlines the future challenges that remain to our understanding of the fascinating post‐translational protein maturation process in more detail.