Cbb3 Terminal Oxidase Research Articles

Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase

The heme‑copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O2 reduction and for proton pumping. This pathway, called the KC-pathway, starts at Glu-49P in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the KC-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pKa of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.

Absence of the cbb3 Terminal Oxidase Reveals an Active Oxygen-Dependent Cyclase Involved in Bacteriochlorophyll Biosynthesis in Rhodobacter sphaeroides.

ABSTRACTThe characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocycle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides. We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to micro-oxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed.IMPORTANCE The formation of the E ring of bacteriochlorophyll pigments is the least well characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high-affinity terminal respiratory oxidase, cytochrome cbb3. We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for this bacterium upon a reduction of oxygen tension.

Genomic characterization of Sinorhizobium meliloti AK21, a wild isolate from the Aral Sea Region.

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti has been widely studied due to its ability to improve crop yields through direct interactions with leguminous plants. S. meliloti AK21 is a wild type strain that forms nodules on Medicago plants in saline and drought conditions in the Aral Sea Region. The aim of this work was to establish the genetic similarities and differences between S. meliloti AK21 and the reference strain S. meliloti 1021. Comparative genome hybridization with the model reference strain S. meliloti 1021 yielded 365 variable genes, grouped into 11 regions in the three main replicons in S. meliloti AK21. The most extensive regions of variability were found in the symbiotic plasmid pSymA, which also contained the largest number of orthologous and polymorphic sequences identified by suppression subtractive hybridization. This procedure identified a large number of divergent sequences and others without homology in the databases, the further investigation of which could provide new insight into the alternative metabolic pathways present in S. meliloti AK21. We identified a plasmid replication module from the repABC replicon family, together with plasmid mobilization-related genes (traG and a VirB9-like protein), which suggest that this indigenous isolate harbors an accessory plasmid. Furthermore, the transcriptomic profiles reflected differences in gene content and regulation between S. meliloti AK21 and S. meliloti 1021 (ExpR and PhoB regulons), but provided evidence for an as yet unknown, alternative mechanism involving activation of the cbb3 terminal oxidase. Finally, phenotypic microarrays characterization revealed a greater versatility of substrate use and chemical degradation than for S. meliloti 1021.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1062-z) contains supplementary material, which is available to authorized users.

R. sphaeroides 에서의 orf282 유전자의 분석과 이들의 기능

The orf282 gene of Rhodobacter sphaeroides is located between the ccoNOQP operon encoding cbb3 terminal oxidase and the fnrL gene encoding an anaerobic activator, FnrL. Its function remains unknown. In an attempt to reveal the function of the orf282 gene, we disrupted the gene by deleting a portion of the orf282 gene and constructed an orf282-knockout mutant. Two FnrL binding sites were found to be located upstream of orf282, and it was demonstrated that orf282 is positively regulated by FnrL. The orf282 gene is not involved in the regulation of spectral complex formation. The cbb3 oxidase activity detected in the orf282 mutant was comparable to that in the wild-type sample, indicating that the orf282 gene is not involved in the regulation of the ccoNOQP operon and the biosynthesis of the cbb3 cytochrome c oxidase. The elevated promoter activity of the nifH and nifA genes, which are the structural genes of nitrogenase and its regulator, respectively, in the orf282 mutant, suggests that the orf282 gene product acts as a negative effector for nifH and nifA expression.

Interacting regulatory networks in the facultative photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1

Regulation of photosynthetic membrane synthesis in Rhodobacter sphaeroides 2.4.1 is dependent on the interactions of numerous regulatory elements, with two of the most important being the cbb(3) terminal oxidase and the PrrBAC two-component regulatory system. Here, we reveal that the cbb(3) terminal oxidase possesses extensive, additional regulatory activities under anaerobic conditions, and that the PrrBAC system is further involved in the regulation of the expression of more than 20% of the R. sphaeroides genome under anaerobic conditions, extending well beyond functions related to redox gene expression.

A purine-related metabolite negatively regulates fixNOQP expression in Sinorhizobium meliloti by modulation of fixK expression.

5-aminoimidazole-4-carboxamide nucleotide (AICAR) is a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli. In this work, the effect of AICAriboside (AICAr), the precursor of AICAR on the expression of the Sinorhizobium meliloti fixNOQP operon encoding the symbiotic terminal oxidase cbb3, was analyzed. AICAr reduced the microaerobic induction levels of fixN-lacZ and fixT-lacZ gene fusions 18- and seven-fold respectively, and both genes were activated by the transcriptional activator FixK. A fixK-lacZ fusion presented 14-fold-reduced induction levels in microaerobic cell cultures in the presence of AICAr. AICAr also reduced three-fold the microaerobic expression levels of the nifA-lacZ fusion, whose expression as well as that of fixK is controlled by the two-component system FixL-FixJ. In contrast, AICAr had no effect on the expression levels of a hemA-lacZ fusion. These data suggest that AICAr prevents fixNOQP induction by the inhibition of fixK transcription.

The cbb3 terminal oxidase of Rhodobacter sphaeroides 2.4.1: structural and functional implications for the regulation of spectral complex formation.

We have previously shown that the flow of reductant through the cbb3 terminal cytochrome c oxidase of Rhodobacter sphaeroides is essential to the repression of photosynthesis (PS) gene expression in the presence of oxygen by inhibiting the functional activity of the Prr two-component activation system. To gain further insight into the role of the cbb3 oxidase and the cognate ccoNOQP operon in the oxygen regulation of PS gene expression, we constructed nonpolar, in-frame deletions within the ccoN and ccoQ genes. Whereas mutations in ccoN, ccoQ, and ccoP resulted in PS gene expression in the presence of oxygen, only the ccoQ mutation showed both the normal flow of reductant through the cbb3 oxidase and the absence of any alteration in the relative levels of spheroidene and spheroidenone, as is observed for those mutations in the cco operon that result in the loss of terminal oxidase activity. Consistent with these findings is the observation that extra copies of the ccoNOQP operon in trans resulted in the decreased formation of both the B800-850 and B875 spectral complexes under anaerobic growth conditions. These results in conjunction with our earlier findings indicate that (1) the flow of reductant through the cbb3 terminal oxidase is a prerequisite to the regulation of PS gene expression by the Prr two-component regulatory system, (2) the CcoQ protein is involved in conveying the signal derived from reductant flow through the cbb3 terminal oxidase to the Prr regulatory pathway, (3) there is reductant flow through this terminal oxidase under anaerobic conditions, and as a result, the activity of the Prr system is still subject to cbb3 regulation, and (4) the acceptor for reductant flow through cbb3 under anaerobic conditions is in whole or in part involved in the conversion of spheroidene to spheroidenone.

Expression of thiamin biosynthetic genes (thiCOGE) and production of symbiotic terminal oxidase cbb3 in Rhizobium etli.

In this paper we report the cloning and sequence analysis of four genes, located on plasmid pb, which are involved in the synthesis of thiamin in Rhizobium etli (thiC, thiO, thiG, and thiE). Two precursors, 4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethylpyrimidine pyrophosphate, are coupled to form thiamin monophosphate, which is then phosphorylated to make thiamin pyrophosphate. The first open reading frame (ORF) product, of 610 residues, has significant homology (69% identity) with the product of thiC from Escherichia coli, which is involved in the synthesis of hydroxymethylpyrimidine. The second ORF product, of 327 residues, is the product of a novel gene denoted thiO. A protein motif involved in flavin adenine dinucleotide binding was found in the amino-terminal part of ThiO; also, residues involved in the catalytic site of D-amino acid oxidases are conserved in ThiO, suggesting that it catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. The third ORF product, of 323 residues, has significant homology (38% identity) with ThiG from E. coli, which is involved in the synthesis of the thiazole. The fourth ORF product, of 204 residues, has significant homology (47% identity) with the product of thiE from E. coli, which is involved in the condensation of hydroxymethylpyrimidine and thiazole. Strain CFN037 is an R. etli mutant induced by a single Tn5mob insertion in the promoter region of the thiCOGE gene cluster. The Tn5mob insertion in CFN037 occurred within a 39-bp region which is highly conserved in all of the thiC promoters analyzed and promotes constitutive expression of thiC. Primer extension analysis showed that thiC transcription in strain CFN037 originates within the Tn5 element. Analysis of c-type protein content and expression of the fixNOQP operon, which codes for the symbiotic terminal oxidase cbb3, revealed that CFN037 produces the cbb3 terminal oxidase. These data show a direct relationship between expression of thiC and production of the cbb3 terminal oxidase. This is consistent with the proposition that a purine-related metabolite, 5-aminoimidazole-4-carboxamide ribonucleotide, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli.

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N',N', tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb3, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c-type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb3 terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-form-aminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.