The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Smith, Andrew T.; Hill, Susan; Anthony, Christopher

doi:10.1099/00221287-136-1-171

Cited by 48 publications

(41 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, in both cases, a d-type terminal oxidase is involved in nitrogen fixation. This enzyme protects nitrogenase against 02 denaturation in A. vinelandii (22) and is required for nitrogen fixation, in the absence of a fermentable carbon source, in K pneumoniae at a 30 nM 02 concentration (17,41). Pure cultures of A. caulinodans display a branched respiratory system: an o-type terminal oxidase is most probably present in addition to the terminal oxidases d and aa3 recently isolated (23).…”

mentioning

confidence: 99%

Functional analysis of the fixNOQP region of Azorhizobium caulinodans

1994

View full text Add to dashboard Cite

The deduced amino acid sequences of four open reading frames identified upstream of thefixGHI region in Azorhizobium caulinodans are very similar to the putative terminal oxidase complex coded by the fixNOQP operons from Rhizobium meliloti and Bradyrhizobium japonicum. The expression of the A. caulinodansfixNOQP genes, which was maximal under microaerobiosis, was positively regulated by FixK and independent of NifA. In contrast to the Fix-phenotype of B.japonicum and R. melilotifixN mutants, anA. caulinodansfixNO-deleted mutant strain retained 50%o of the nitrogenase activity of the wild type in the symbiotic state. In addition, the nitrogenase activity was scarcely reduced under free-living conditions. Analysis of membrane fractions of A.caulinodans wild-type and mutant strains suggests that the fixNOQP region encodes two proteins with covalently bound hemes, tentatively assigned tofixO andfixP. Spectral analysis showed a large decrease in the c-type cytochrome content of thefixN mutant compared with the wild type. These results provide evidence for the involvement of FixNOQP proteins in a respiratory process. The partial impairment in nitrogen fixation of thefixN mutant in planta may be due to the activity of an alternative terminal oxidase compensating for the loss of the oxidase complex encoded byfixNOQP.

show abstract

mentioning

confidence: 99%

Functional analysis of the fixNOQP region of Azorhizobium caulinodans

1994

View full text Add to dashboard Cite

show abstract

“…To fix N2 in air, Azotobacter spp. require cytd oxidase (22), which might also mitigate oxidative stress (19,22,38). The physiological importance of cyto remains unclear.…”

mentioning

confidence: 99%

“…The physiological importance of cyto remains unclear. Klebsiella pneumoniae, a facultative anaerobe, fixes N2 in microaerobiosis and also employs a cytd quinol oxidase (19,38). Symbiotic N2-fixing rhizobia, obligate aerobes, exhibit multiple cytochrome oxidases in culture and in planta.…”

mentioning

confidence: 99%

Azorhizobium caulinodans respires with at least four terminal oxidases

Kitts

Ludwig

1994

J Bacteriol

View full text Add to dashboard Cite

In culture, Azorhizobium caulinodans used at least four terminal oxidases, cytochrome aa3 (cytaa3), cytd, cyto, and a second a-type cytochrome, which together mediated general, respiratory electron (e-) transport to 02. To genetically dissect physiological roles for these various terminal oxidases, corresponding Azorhizobium apocytochrome genes were cloned, and three cytaa3 mutants, a cytd mutant, and a cytaa3, cytd double mutant were constructed by reverse genetics. These cytochrome oxidase mutants were tested for growth, oxidase activities, and N2 fixation properties both in culture and in symbiosis with the host plant Sesbania rostrata. The cytaa3 mutants grew normally, fixed N2 normally, and remained fully able to oxidize general respiratory edonors (NADH, succinate) which utilize a cytc-dependent oxidase. By difference spectroscopy, a second, a-type cytochrome was detected in the cytaa3 mutants. This alternative a-type cytochrome (Amax = 610 nm) was also present in the wild type but was masked by bona fide cytaa3 (Amax = 605 nm). In late exponential-phase cultures, the cytaa3 mutants induced a new, membrane-bound, CO-binding cytc550, which also might serve as a cytc oxidase (a fifth terminal oxidase). The cloned Azorhizobium cytaa3 genes were strongly expressed during exponential growth but were deactivated prior to onset of stationary phase. Azorhizobium cytd mutants showed 401% lower N2 fixation rates in culture and in planta, but aerobic growth rates were wild type. The cytaa3, cytd double mutant showed 70%6 lower N2 fixation rates in planta. Pleiotropic cytc mutants were isolated by screening for strains unable to use N,N,N',N'-tetramethyl-p-phenylenediamine as a respiratory e -donor. These mutants synthesized no detectable cytc, excreted coproporphyrin, grew normally in aerobic minimal medium, grew poorly in rich medium, and fixed N2 poorly both in culture and in planta. Therefore, while aerobic growth was sustained by quinol oxidases alone, N2 fixation required cytc oxidase activities. Assuming that the terminal oxidases function as do their homologs in other bacteria, Azorhizobium respiration simultaneously employs both quinol and cytc oxidases. Because Azorhizobium terminal oxidase mutants were able to reformulate their terminal oxidase mix and grow more or less normally in aerobic culture, these terminal oxidases are somewhat degenerate. Its extensive terminal oxidase repertoire might allowAzorhizobium spp. to flourish in wide-ranging 02 environments.As biological N2 fixation is extremely 02 sensitive, N2-fixing organisms have evolved 02 detoxification and/or avoidance mechanisms. Azotobacter spp., free-living aerobes, elaborate cells with very high total membrane surface area and, consequently, very high concentrations of respiratory electron (e-) transport complexes. For respiration, Azotobacter spp. employ cytochrome d (cytd) and cytochrome o (cyto) quinol oxidases but not cytc oxidases (20,44). To fix N2 in air, Azotobacter spp. require cytd oxidase (22), which might also mitigate oxidative ...

show abstract

“…Steady state kinetics in ubiquinol oxidases has been interpreted as a simple Michaelis-Menten type. The K m values estimated for Q 1 H 2 are usually ϳ0.1-0.2 mM (5,22,43,45,47,48,52), and the K m value for Q 2 H 2 has been reported to be 0.05 mM for the E. coli enzyme (22). Because of auto-oxidation of low potential quinols, the K m values are not reported for menaquinol oxidases.…”

Section: Discussionmentioning

confidence: 99%

“…Cytochrome bd has been isolated as ubiquinol oxidase from ␥-proteobacteria including E. coli (5,(43)(44)(45)(46)(47)(48) and as menaquinol oxidase from Gram-positive bacteria (49 -51). Steady state kinetics in ubiquinol oxidases has been interpreted as a simple Michaelis-Menten type.…”

Section: Discussionmentioning

confidence: 99%

Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

Matsumoto

Murai

Fujita

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross- Cytochrome bd (CydAB) is a heterodimeric ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is predominantly expressed under microaerophilic growth conditions (see Refs. 1-3 for reviews). It catalyzes dioxygen reduction with two molecules of ubiquinol-8 (Q 8 H 2 ), 2 leading to the release of four protons from quinols to the periplasm. Through a putative proton channel, four protons used for dioxygen reduction are taken up from the cytoplasm and delivered to the dioxygen reduction site at the periplasmic side of the cytoplasmic membrane (4). During dioxygen reduction, cytochrome bd generates an electrochemical proton gradient (⌬pH and membrane potential) across the membrane through apparent transmembrane movement of four chemical protons (5-7). In contrast to cytochrome bo, an alternative oxidase under highly aerated growth conditions, cytochrome bd has no proton pumping activity and does not belong to the heme-copper terminal oxidase superfamily.On the basis of spectroscopic and ligand binding studies, three distinct redox metal centers have been identified as heme b 558 , heme b 595 , and heme d (see Ref. 8 for a review). Unlike cytochrome bo, cytochrome bd does not contain a tightly bound Q 8 . Heme b 558 is a low spin protoheme IX and is ligated by I-His 186 (helix V) and I-Met 393 (helix VII) of subunit I (CydA) (9). Heme b 595 is a high spin protoheme IX bound to I-His 19 (helix I) of subunit I (9) and mediates electron transfer from heme b 558 to heme d, where dioxygen is reduced to water (10 -13). Heme d is a high spin chlorin bound to an unidentified nitrogenous ligand (14 -16) and forms a di-heme binuclear center with heme b 595 (16,17). Topological analysis suggests that all of the hemes are located at the periplasmic end of transmembrane helices (4).In loop VI/VII (Q-loop) of subunit I, binding of monoclonal antibodies to 252 KLAAIEAEWET 262 (18,19) and proteolytic cleavage with trypsin at I-Tyr 290 or chymotrypsin at I-Arg 298 (20, 21) suppressed ubiquinol oxidase activity. Photoaffinity labeling studies with 2-methyl-3-azido-5-methoxy-6-(3,7-dimethyl-[ 3 H]octyl)-1,4-benzoquinone ([ 3 H]3-azido-2-methyl-5-methoxy-BQ 2s ) indicate the presence of the quinol oxidation site in subunit I (22). These results suggest that periplasmic loop VI/VII in subunit I is involved in the ubiquinol oxidation site. Inhibitor binding studies indicated the close proximity ...

show abstract

The purification, characterization and role of the d-type cytochrome oxidase of Klebsiella pneumoniae during nitrogen fixation

Cited by 48 publications

References 30 publications

Functional analysis of the fixNOQP region of Azorhizobium caulinodans

Functional analysis of the fixNOQP region of Azorhizobium caulinodans

Azorhizobium caulinodans respires with at least four terminal oxidases

Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

Contact Info

Product

Resources

About