Yoshihiro Sambongi

Contrasting routes of c-type cytochrome assembly in mitochondria, chloroplasts and bacteria

The biogenesis of bacterial c-type cytochromes generally involves many gene products--some of which may also have roles in other processes--and their interaction with the disulphide-bond-forming system of the bacterial periplasm. However, in some bacteria a simpler process appears to operate that might be related to the formation of c-type cytochromes in thylakoids of photosynthetic cells. The corresponding process in fungal mitochondria is distinct.

Sensing of cadmium and copper ions by externally exposed ADL, ASE, and ASH neurons elicits avoidance response in Caenorhabditis elegans.

We developed a quantitative assay for Caenorhabditis elegans avoidance behavior. This was then used to demonstrate that the worm moved away from toxic concentrations of Cd2+ and Cu2+, but not Ni2+, all ions that prevented development from larval to adult stages. Mutants that have structural defects in ciliated neurons (che-2 and osm-3) as well as worms with three laser-operated neurons (ADL, ASE, and ASH), showed no avoidance behavior from Cd2+ and Cu2+. These results suggest that the avoidance from Cd2+ and Cu2+ are mediated through multiple neural pathways including ADL, ASE, and ASH neurons. We hypothesize that the three sensing neurons provide increased accuracy of the sensory response and a survival advantage in the natural environment of the worm.

Analysis of functional domains of Wilson disease protein (ATP7B) in Saccharomyces cerevisiae

Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver. The ATP7B gene, which encodes a copper transporting P‐type ATPase, is defective in patients with Wilson disease. To investigate the function of ATP7B, wild type or mutated ATP7B cDNA was introduced into a yeast strain lacking the CCC2 gene (Δccc2), the yeast homologue of ATP7B. Wild type and the H1069Q mutant could rescue Δccc2, however, the N1270S mutant could not, reflecting phenotypic variability of Wilson disease. In addition, the mutant containing only the sixth copper binding domain could rescue Δccc2, indicating its functional importance.

Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase

The structural gene, nrfA, for cytochrome C552, which is the terminal reductase of the formate‐dependent pathway for nitrite reduction to ammonia, has been located at co‐ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c‐type cytochrome with a predicted Mr for the unprocessed product of 53788. Cleavage of the putative signal peptide at Ala‐26 would result in a mature, periplasmic cytochrome of Mr 50580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS‐polyacryla‐mide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome C552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in‐phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In‐phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping.

Stabilization of Pseudomonas aeruginosa Cytochromec 551 by Systematic Amino Acid Substitutions Based on the Structure of Thermophilic Hydrogenobacter thermophilus Cytochrome c 552

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochromec 551 (PA c 551) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c 551 with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochromec 552 (HT c 552), which has similar main chain folding to PA c 551, but is more stable to heat. Thermodynamic properties of PAc 551 with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c 551 with HTc 552. In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c 552. We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PAc 551 can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c 552. The higher stability of HT c 552 may in part be attributed to some of these substitutions.

Specific thiol compounds complement deficiency in c-type cytochrome biogenesis in Escherichia coli carrying a mutation in a membrane-bound disulphide isomerase-like protein.

Escherichia coli JCB606 carries a mutation in the dipZ gene, known to code for a disulphide isomerase‐like protein, with the consequence that holo forms of neither exogenous nor endogenous c‐type cytochromes are synthesised. This failure has been overcome by adding compounds containing thiol groups to the growth medium. Only l‐Cysteine and 2‐mercaptoethane sulphonic acid were effective, suggesting a (stereo)specific binding site that could be occupied by these compounds in the absence of the catalytic domain of DipZ.

Synthesis of holo Paracoccus denitrificans cytochrome c550 requires targeting to the periplasm whereas that of holo Hydrogenobacter thermophilus cytochrome c552 does not: Implications for c-type cytochrome biogenesis

Expression from a plasmid of the complete gene, including the codons for the N‐terminal periplasmic targeting signal, for cytochrome c 550 of Paracoccus denitrificans led to the formation of the holo protein in the periplasms of both P. denitrificans and Escherichia coli. Expression of the gene from which the region coding for the targeting signal had been specifically deleted resulted in formation of apo‐protein in the cytoplasms of both organisms. These findings are consistent with haem attachment occurring in the periplasm. In contrast, the formation of holo cytochrome c 552 from Hydrogenobacter thermophilus following expression of the gene lacking the periplasmic targeting sequence in either P. denitrificans or E. coli is attributed to spontaneous cytoplasmic attachment of haem to the thermostable protein.

Mutants of Escherichia coli lacking disulphide oxdoreductases DsbA and DsbB cannot synthesise an exogenous monohaem c-type cytochrome except in the presence of disulphide compounds

Absence through mutation of two proteins involved in periplasmic disulphide bond formation, DsbA and DsbB, results in failure of anaerobically grown Escherichia coli to synthesise the holo forms of either its endogenous c‐type cytochrome nitrite reductase or exogenous cytochrome c 550 from Paracoccus denitrificans. The synthesis of both cytochromes can be restored to the mutants by inclusion in the growth media of compounds containing disulphide bonds, e.g., the oxidised form of glutathione. The results suggest that the attachment of haem to the CXXCH motif of a periplasmic c‐type cytochrome may be preceeded by the formation of one or more intra‐ or intermolecular disulphide bonds involving the cysteine residues of this motif.

Alteration of haem‐attachment and signal‐cleavage sites for Paracoccus denitrificans cytochrome c550 probes pathway of c‐type cytochrome biogenesis in Escherichia coli

Paracoccus denitrificans cytochrome c550 is expressed as a periplasmic holo‐protein in Escherichia coli; amino acid substitutions of cysteine residues in the haembinding motif (Cys‐X‐X‐Cys‐His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions ‐3 to ‐1 in the native signal‐cleavage site were deleted, or alanine at ‐1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp‐Glu‐Asp, a membrane‐associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo‐cytochrome c550 export to the periplasm; (ii) haem cannot attach to apocytochrome c550 when attached to the cytoplasmic membrane, suggesting that signal‐sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N‐terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso‐1‐cytochrome c was expressed as a holo‐protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome c550, indicating that the E. coli cellular apparatus for the c‐type cytochrome biogenesis has a broad substrate specificity.

Biological nano motor, ATP synthase FoF1: from catalysis to γϵc10–12 subunit assembly rotation

Abstract Proton translocating ATPase (ATP synthase), a chemiosmotic enzyme, synthesizes ATP from ADP and phosphate coupling with the electrochemical ion gradient across the membrane. This enzyme has been studied extensively by combined genetic, biochemical and biophysical approaches. Such studies revealed a unique mechanism which transforms an electrochemical ion gradient into chemical energy through the rotation of a subunit assembly. Thus, this enzyme can be defined as a nano motor capable of coupling a chemical reaction and ion translocation, or more simply, as a protein complex carrying out rotational catalysis. In this article, we briefly discuss our recent work, emphasizing the rotation of subunit assembly (γϵ c 10–12 ) which is formed from peripheral and intrinsic membrane subunits.