Paracoccus denitrificans cytochrome c1 gene replacement mutants

Abstract

We describe the construction and characterization of Essentially three features have made the gram-negative soil bacterium Paracoccus denitrificans an interesting organism for bioenergetic research and, in particular, a model organism for the mitochondrial electron transport. (i) Its close relationship to present-day mitochondria, indicated by a large number of mostly physiological and bioenergetic arguments, prompted John and Whatley (23,24) to assign this bacterium a hypothetical precursor role in endosymbiotic organelle development; this early assumption of a close evolutionary homology has recently been confirmed by more direct evidence on phylogenetic grounds (59). (ii) At least two respiratory complexes, cytochrome c reductase (see below) and cytochrome c oxidase (17,30,32) isolated from this bacterium in a functional state, have basic enzymatic properties almost indistinguishable from those of their mitochondrial counterparts, but are far less complex in their structure. (iii) Due to the availability of the genes for their subunits, these enzymes are amenable to site-specific mutagenesis experiments to study stucture-function relationships and thus increase our knowledge on basic electron transport and energy transduction mechanisms both in bacteria and in mitochondria.The cytochrome bc, complex (complex III; ubiquinol: cytochrome c oxidoreductase [EC 1.10.2.2]) is an obligate component in the electron transport chain of mitochondria and, in an analogous form, of photosynthetic organisms (19,20,43,58). Moreover, several bacterial species have recently been shown to contain a functionally homologous complex, notably Rhodobacter species (8,13,14) and Paracoccus denitrificans (4, 33, 60). Three different subunits carrying the redox centers of this integral membrane enzyme, cytochromes b and cl and the so-called Rieske protein with its iron-sulfur center (FeS), are common to all bc, complexes. Whereas the well-studied enzyme complexes from mitochondria typically contain a large number of additional polypeptides of uncertain function, the bacterial enzymes mentioned above are composed of only the three essential redox center subunits. However, at least for Para-* Corresponding author. coccus spp., full enzymatic competence of this structurally simple complex has been reported in a reconstituted system, for both its electron transport and energy transduction capacity (60, 61). Molecular details of these two coupled processes have not been elucidated in mitochondrial bc1 complexes, but hypotheses for structure-function relationships as well as tentative functional assignments for certain residues have been made (10,22,38,45,52,57). Such assumptions can be tested in bacteria by performing sitedirected mutagenesis on the isolated genes, followed by insertion into a suitable host to obtain expression of the redox complex. In Paracoccus spp., the genes for the bc1 subunits have been cloned and sequenced (27), and, as in Rhodobacter spp. (8, 13), have been shown to be organized in an operon structure, subsequent...

NMR assignments and relaxation studies of Thiobacillus versutus ferrocytochrome c‐550 indicate the presence of a highly mobile 13‐residues long C‐terminal tail

“…denitri$cans (Pd2131) in which the endogenous gene for cytochrome c-550 had been deleted ( Van Spanning et al, 1991) was used as host for expression. The gene encoding cytochrome c-550 of I: versutus and its putative promoter region were introduced in the host on a stable, multicopy plasmid (pEG400) (Gerhus et al, 1990). With this strain, a yield was obtained of IO mg of cytochrome c-550 per liter, when cultured on rich medium.…”

“…The gene for 7: versutus cytochrome c-550 and its putative promoter region (Sph I-Sph I restriction fragment of pMU8) (Ubbink et al, 1992) were inserted in vector pEG400 (Gerhus et al, 1990) and conjugated from E. coli TGl to P. denitriJicans Pd2131 via triple conjugation using E. coli HB101/pRK2020 (De Gier et al, 1994). In P. denitrifcans Pd2 13 1, the native gene for cytochrome c-550 has been deleted ( Van Spanning et al, 1991).…”

Mutation of Arg-54 Strongly Influences Heme Composition and Rate and Directionality of Electron Transfer in Paracoccus denitrificans Cytochrome c Oxidase

“…Bacteria were grown in succinate medium (26) at 32°C, and cells were harvested at the late exponential phase. Bacterial membranes were isolated as described (27) and solubilized with n-dodecyl-␤-D-maltopyranoside (28). Cytochrome c oxidase was then purified by streptavidin affinity chromatography as described by Kleymann et al (28), using an engineered streptavidin (29).…”

Perturbation of the CuA Site in Cytochrome‐c Oxidase of Paracoccus denitrificans by Replacement of Met227 with Isoleucine

“…Cells were grown on succinate medium [19] including 25 mg/l streptomycin sulfate and a decreased manganese concentration of 0.5 !AM to avoid the strong interfering manganese signal in the EPR spectra 1201. Metnbranes were prepared as described in [16] and solubilized with the detergent n-dodecyl-/3-r)-maltoside. Protein purification was according to 121 I including 1 mM EDTA in all buffers.…”