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In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative electron carriers from the cytochrome bc(1) complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c(550). The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c(550) or both these proteins. We concluded that pseudoazurin and cytochrome c(550) are the alternative electron mediator proteins between the cytochrome bc(1) complex and the cytochrome cd(1)-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

Each of the Paracoccus denitrificans genes in the c-type cytochrome biogenesis gene cluster ccmABCDG, plus the two flanking genes ORF117 and hisH, were individually disrupted by omega insertion. Resultant phenotypes were restored to the wild-type by complementation from a set of plasmids. All of the ccm genes, but neither ORF117 nor hisH, were required for c-type cytochrome biogenesis; only ccmG was also implicated in the biosynthesis of cytochrome aa3. Disruption of ccmC or ccmG resulted in failure to grow on rich media, but disruption of ccmA, ccmB or ccmD did not. The ccmC mutant, but not the ccmA, ccmB or ccmD mutants, also exhibited the increased sensitivity to growth inhibition by oxidized thiol compounds previously observed for the ccmG mutant. In contrast to the ccmG mutant, however, growth of the ccmC mutant on rich media could not be restored by DTT. Siderophore biosynthesis and/or secretion by P. denitrificans was also attenuated by disruption of ccmC and ccmG but not of ccmA, ccmB or ccmD. These results indicate that CcmC can function independently of CcmA, CcmB and CcmD despite other evidence that these gene products form an ATP-binding cassette (ABC)-type-transporter with the subunit composition (CcmA)2-CcmB-CcmC or (CcmA)2-CcmB-CcmC-CcmD, and also suggest a possible link between the functions of CcmC and CcmG.

Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein-disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG-PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c-type cytochromes, but also loss of spectroscopically detectable cytochrome aa3, cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa3 and cytochrome c oxidase activity, although it did not restore c-type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein-disulphide bonds in certain protein substrates including c-type cytochrome polypeptides and/or polypeptides involved in c-type cytochrome biogenesis.

The periplasmic cytochrome c550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.

By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, moxG, and moxI gene products of Methylobacterium extorquens AM1. Directly downstream from these three genes, a new mox gene was identified. The gene is designated moxR. By using the suicide vector pGRPd1, the moxJ, moxG, and moxR genes were inactivated by the insertion of a kanamycin resistance gene. Subsequently, suicide vector pRVS1 was used to replace the marker genes in moxJ and moxG for unmarked deletions made in vitro. As a result, the three insertion strains as well as the two unmarked mutant strains were unable to grow on methanol, even in the presence of pyrroloquinoline quinone. Growth on succinate and on methylamine was not affected. In all five mutant strains, synthesis of the large subunit of methanol dehydrogenase and of inducible cytochrome c553i was observed. The moxJ and moxG insertion mutant strains were unable to synthesize both the cytochrome c551i and the small subunit of methanol dehydrogenase, and this lack of synthesis was attended by the loss of methanol dehydrogenase activity. The moxJ deletion mutant strain partly synthesized the latter two proteins, cytochrome c551i. Partial synthesis of the small subunit of methanol dehydrogenase observed with the latter strain was attended by a corresponding extent of methanol dehydrogenase activity. The moxR insertion mutant strain was shown to synthesize cytochrome c551i as well as the large and small subunits of methanol dehydrogenase, but no methanol dehydrogenase activity was observed. The results show that periplasmic cytochrome c551i is the moxG gene product and the natural electron acceptor of methanol dehydrogenase in P. denitrificans. In contrast to earlier suggestions, this cytochrome was found to be different from membrane-bound cytochrome c552. In addition, it is demonstrated that moxI encodes the small subunit of methanol dehydrogenase. It is suggested that MoxJ is involved in the assemblage of active methanol dehydrogenase in the periplasm and, in addition, that MoxR is involved in the regulation of formation of active methanol dehydrogenase.

By using synthetic oligonucleotides, the gene encoding soluble cytochrome c550 was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the gene was determined, and the deduced amino acid sequence of the mature protein was found to be similar to the primary structure of purified cytochrome c550 except for the presence of seven additional amino acid residues at the C terminus. At the N terminus of the primary structure was found an additional stretch of 19 amino acid residues that had the typical features of the signal sequence of the cytochrome. Comparison of the nucleotide sequences of the upstream regions of the P. denitrificans cytochrome c550 gene and bc1 operon revealed three regions with a distinct organization that showed strong similarity. Downstream of the c550 gene was found part of another gene, the deduced amino acid sequence of which showed strong homology with subunit 1 of the cytochrome aa3 oxidase. For gene replacement experiments, the suicide vector pGRPd1 was constructed. The cytochrome c550 gene was inactivated by insertion of a kanamycin resistance gene, and the mutated gene was cloned into this vector. Recombination with the wild-type gene resulted in a mutant strain with an inactivated cytochrome gene. Isolated mutant strains were unable to synthesize the soluble cytochrome, as judged by spectrum analysis and analysis of periplasmic proteins by gel electrophoresis and heme staining. The mutation resulted in a 14% decrease in the growth yield during aerobic heterotrophic growth and in a 40% decrease in the maximum specific growth rate during growth on methylamine. Furthermore, a longer lag phase was observed under both growth conditions. The mutation had no effect on growth yield, maximum specific growth rate, and duration of the lag phase during anaerobic growth in the presence of nitrate. In addition, there was no accumulation of nitrite and nitrous oxide.

Soluble c-type cytochromes were partially purified from Paracoccus denitrificans cells grown in succinate- and methanol-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. Five c types could be distinguished with the following apparent molecular masses, absorption maxima and midpoint potentials. (a) 9.2 kDa, 549 nm and +190 mV; (b) 14 kDa, 549 nm and +227 mV; (c) 22 kDa, 552 nm and +190 mV; (d) 30 kDa, 552.7 nm and +160 mV; (e) 45 kDa, a dihaem: 555 nm, +128 mV and 551 nm, -163 mV. The 14-kDa polypeptide was present under all growth conditions examined and most probably is the already well characterized cytochrome c550. In methanol-limited grown cells three additional cytochromes were found, the 9.2-kDa, 22-kDa and 30-kDa ones. Under oxygen-limited conditions the 45-kDa and under anaerobic growth conditions small quantities of the 30-kDa and 45-kDa cytochromes c were present. Based on the apparent molecular masses the 14-kDa, 22-kDa, 30-kDa and 45-kDa cytochromes may also be present in membrane-fractions.

An apo form of cytochrome cd1 (nitrite reductase) of Paracoccus denitrificans has been detected immunologically in the periplasm of a mutant that lacks all c-type cytochromes. A method for the preparation of apo-nitrite reductase (lacking both c- and d-type haem) from the holoenzyme of wild-type cells has been developed. The apoprotein synthesized by the mutant is indistinguishable from the chemically prepared apoprotein in respect of: (i) subunit molecular weight; (ii) formation of a homodimer; (iii) properties on anion exchange chromatography. The holoenzyme has similar properties in respect of (i) and (ii) but behaves differently during anion exchange. A suggested mode of assembly of cytochrome cd1 is translocation into the periplasm of a precursor polypeptide, maturation by a signal peptidase to give an apoprotein identical to that prepared chemically from the holoenzyme, followed by insertion of c-type and d-type haem in an as yet unknown order.