PubMed Health⌕ Search

Biomedical subjects

Robert K Poole

Publications and source records attributed to Robert K Poole.

At least 37 records · Page 2Linked to original sources
PubMed2003

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp.

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.

Dihydropteridine Reductase↗
PubMed2003

Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis.

Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well understood, contains at least 7 iron-acquisition systems encoded by 35 iron-repressed genes. This Fe-dependent repression is mediated by a transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other processes such as iron storage, the Tricarboxylic Acid Cycle, pathogenicity, and redox-stress resistance. Our macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways. Interestingly, a large group of energy metabolism genes was found to be iron and Fur induced. Many of these genes encode iron-rich respiratory complexes. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E. coli can modulate its iron requirements. Analysis of 55Fe-labeled E. coli proteins revealed a marked decrease in iron-protein composition for the fur mutant, and visible and EPR spectroscopy showed major reductions in cytochrome b and d levels, and in iron-sulfur cluster contents for the chelator-treated wild-type and/or fur mutant, correlating well with the array and quantitative RT-PCR data. In combination, the results provide compelling evidence for the regulation of intracellular iron consumption by the Fe2+-Fur complex.

Biological Transport↗
PubMed2003

Microbial globins.

Globins are an ancient and diverse superfamily of proteins. The globins of microorganisms were relatively ignored for many decades after their discovery by Warburg in the 1930s and rediscovery by Keilin in the 1950s. The relatively recent focus on them has been fuelled by recognition of their structural diversity and fine-tuning to fulfill (probably) discrete functions but particularly by the finding that a major role of certain globins is in protection from the stresses caused by exposure to nitric oxide (NO)--itself a molecule that has attracted intense curiosity recently. At least three classes of microbial globin are recognised, all having features of the classical globin protein fold. The first class is typified by the myoglobin-like haemprotein Vgb from the bacterium Vitreoscilla, which has attracted considerable attention because of its ability to improve growth and metabolism for biotechnological gain in a variety of host cells, even though its physiological function is not fully understood. The truncated globins are widely distributed in bacteria, microbial eukaryotes as well as plants and are characterised by being 20-40 residues shorter than Vgb. The polypeptide is folded into a two-over-two helical structure while retaining the essential features of the globin superfamily. Roles in oxygen and NO metabolism have been proposed. The third and best understood class comprises the flavohaemoglobins, which were first discovered and partly characterised in yeast. These are distinguished by the presence of an additional domain with binding sites for FAD and NAD(P)H. Widely distributed in bacteria, these proteins undoubtedly confer protection from NO and nitrosative stresses, probably by direct consumption of NO. However, a bewildering array of enzymatic capabilities and the presence of an active site in the haem pocket reminiscent of peroxidases hint at other functions. A full understanding of microbial globins promises advances in controlling the interactions of pathogenic bacteria with their animal and plant hosts, and manipulations of microbial oxygen transfer with biotechnological applications.

Amino Acid Sequence↗
PubMed2003

Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions.

The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by live bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by live bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia.

Adsorption↗
PubMed2003

The paradoxical cyanide-stimulated respiration of Zymomonas mobilis: cyanide sensitivity of alcohol dehydrogenase (ADH II).

The respiratory inhibitor cyanide stimulates growth of the ethanologenic bacterium Zymomonas mobilis, perhaps by diverting reducing equivalents from respiration to ethanol synthesis, thereby minimizing accumulation of toxic acetaldehyde. This study sought to identify cyanide-sensitive components of respiration. In aerobically grown, permeabilized Z. mobilis cells, addition of 200 microM cyanide caused gradual inhibition of ADH II, the iron-containing alcohol dehydrogenase isoenzyme, which, in aerobic cultures, might be oxidizing ethanol and supplying NADH to the respiratory chain. In membrane preparations, NADH oxidase was inhibited more rapidly, but to a lesser extent, than ADH II. The time-course of inhibition of whole-cell respiration resembled that of NADH oxidase, yet the inhibition was almost complete, and was accompanied by an increase of intracellular NADH concentration. Cyanide did not significantly affect the activity of ADH I, the zinc-containing alcohol dehydrogenase isoenzyme. When an aerobic batch culture was grown in the presence of 200 microM cyanide, cyanide-resistant ADH II activity was observed, its appearance correlating with the onset of respiration. It is concluded that the membrane-associated respiratory chain, but not ADH II, is responsible for the whole-cell cyanide sensitivity, while the cyanide-resistant ADH II is needed for respiration in the presence of cyanide, and represents an adaptive response of Z. mobilis to cyanide, analogous to the induction of alternative terminal oxidases in other bacteria.

Aerobiosis↗
PubMed2002

Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly.

Assembly of Escherichia coli cytochrome bd and periplasmic cytochromes requires the ATP-binding cassette transporter CydDC, whose substrate is unknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-type and cydD mutant strains revealed that the latter was deficient in several periplasmic transport binding proteins, but no single major protein was missing in the cydD periplasm. Instead, CydDC exports from cytoplasm to periplasm the amino acid cysteine, demonstrated using everted membrane vesicles that transported radiolabeled cysteine inward in an ATP-dependent, uncoupler-independent manner. New pleiotropic cydD phenotypes are reported, including sensitivity to benzylpenicillin and dithiothreitol, and loss of motility, consistent with periplasmic defects in disulfide bond formation. Exogenous cysteine reversed these phenotypes and affected levels of periplasmic c-type cytochromes in cydD and wild-type strains but did not restore cytochrome d. Consistent with CydDC being a cysteine exporter, cydD mutant growth was hypersensitive to high cysteine concentrations and accumulated higher cytoplasmic cysteine levels, as did a mutant defective in orf299, encoding a transporter of the major facilitator superfamily. A cydD orf299 double mutant was extremely cysteine-sensitive and had higher cytoplasmic cysteine levels, whereas CydDC overexpression conferred resistance to high extracellular cysteine concentrations. We propose that CydDC exports cysteine, crucial for redox homeostasis in the periplasm.

ATP-Binding Cassette Transporters↗
PubMed2002

A novel haem compound accumulated in Escherichia coli overexpressing the cydDC operon, encoding an ABC-type transporter required for cytochrome assembly.

cydDC genes encode a heterodimeric ABC transporter required for assembly of the membrane-bound cytochrome bd quinol oxidase and periplasmic cytochromes. Here, we demonstrate that overexpression of functional cydDC genes on a multicopy plasmid results in elevated levels of cytochromes b and d, but most notably formation in anaerobically grown cells of a novel haem-containing component P-574. The pigment has a distinctive absorbance at 574-579 nm and 448 nm in reduced minus oxidised spectra and renders over-producing cells reddish in colour. The highest levels of P-574 were observed in mutants (cydAB) in the structural genes for the polypeptides of cytochrome bd. P-574 is labile; its spectral signal is reduced in cells that are frozen-thawed or subjected to mechanical disruption. P-574 was not detected in cytoplasmic or periplasmic fractions and was predominantly associated with the cell membrane. P-574 did not bind CO or cyanide. Production of P-574 was dependent on haem biosynthesis indicating that it is a haem-containing molecule or derived from haem biosynthesis. These findings suggest that P-574 may result from association of a haem compound with overexpressed transporter subunits, but not with oxidase subunits, and are consistent with an intimate link between the transporter and haem processing during oxidase assembly.

ATP-Binding Cassette Transporters↗
PubMed2002

Nitric oxide releases intracellular zinc from prokaryotic metallothionein in Escherichia coli.

Nitric oxide (NO) has a broad spectrum of signalling and regulatory functions and multiple molecular targets. Recently, the intrabacterial toxicity of NO and mechanisms for NO resistance have been intensively investigated. Here we report for the first time that NO elicits release of zinc from a bacterial protein. Using the zinc-responsive expression of zntA (encoding a Zn-exporting P-type ATPase) fused to lacZ, i.e. Phi(zntA-lacZ), to monitor intracellular zinc, and SmtA (the Synechococcus metallothionein) as zinc store, we have shown that the NO donors NOC-5 and NOC-7 elicit zinc ejection. No increase in Phi(zntA-lacZ) activity was observed in a zntR mutant, indicating the specificity of the zntA promoter response to zinc ions.

Adenosine Triphosphatases↗
PubMed2002

NO sensing by FNR: regulation of the Escherichia coli NO-detoxifying flavohaemoglobin, Hmp.

Nitric oxide (NO) is a signalling and defence molecule of major importance in biology. The flavohaemoglobin Hmp of Escherichia coli is involved in protective responses to NO. Because hmp gene transcription is repressed by the O(2)-responsive regulator FNR, we investigated whether FNR also senses NO. The [4Fe-4S](2+) cluster of FNR is oxygen labile and controls protein dimerization and site-specific DNA binding. NO reacts anaerobically with the Fe-S cluster of purified FNR, generating spectral changes consistent with formation of a dinitrosyl-iron-cysteine complex. NO-inactivated FNR can be reconstituted, suggesting physiological relevance. FNR binds at an FNR box within the hmp promoter (P(hmp)). FNR samples inactivated by either O(2) or NO bind specifically to P(hmp), but with lower affinity. Dose-dependent up-regulation of P(hmp) in vivo by NO concentrations of pathophysiological relevance is abolished by fnr mutation, and NO also modulates expression from model FNR-regulated promoters. Thus, FNR can respond to not only O(2), but also NO, with major implications for global gene regulation in bacteria. We propose an NO-mediated mechanism of hmp regulation by which E.coli responds to NO challenge.

Anaerobiosis↗
PubMed2002

Flavohemoglobin Hmp protects Salmonella enterica serovar typhimurium from nitric oxide-related killing by human macrophages.

Survival of macrophage microbicidal activity is a prerequisite for invasive disease caused by the enteric pathogen Salmonella enterica serovar Typhimurium. Flavohemoglobins, such as those of Escherichia coli, Salmonella, and yeast, play vital roles in protection of these microorganisms in vitro from nitric oxide (NO) and nitrosative stress. A Salmonella hmp mutant defective in flavohemoglobin (Hmp) synthesis exhibits growth that is hypersensitive to nitrosating agents. We found that respiration of this mutant exhibited increased inhibition by NO, whereas wild-type cells pregrown with sodium nitroprusside or S-nitrosoglutathione showed enhanced tolerance of NO. Most significantly, hmp mutants internalized by primary human peripheral monocyte-derived macrophages survived phagocytosis relatively poorly compared with similarly bound and internalized wild-type cells. That the enhanced sensitivity to macrophage microbicidal activity is due primarily to the failure of Salmonella to detoxify NO was suggested by the ability of L-N(G)-monomethyl arginine-an inhibitor of NO synthase-to eliminate the difference in killing between wild-type and hmp mutant Salmonella cells. These observations suggest that Salmonella Hmp contributes to protection from NO-mediated inhibition by human macrophages.

Bacterial Adhesion↗
PubMed2000

Oxidase and periplasmic cytochrome assembly in Escherichia coli K-12: CydDC and CcmAB are not required for haem-membrane association.

The mechanism(s) that bacteria use to transport haem into and across the cytoplasmic membrane to complete the assembly of periplasmic cytochromes is unknown. The authors have tested directly the role(s) of two ATP-binding cassette (ABC) transporters - the cydDC and ccmAB gene products - in Escherichia coli by measuring haem uptake in everted (inside-out) membrane vesicles. If haem is exported to the periplasm in vivo, the same process should result in active accumulation in such everted vesicles. [14C]Haemin (chloride) with bovine serum albumin (BSA) as a carrier protein was accumulated in intact everted membrane vesicles by an energy-independent mechanism. The kinetics of this process were biphasic: rapid uptake/binding was followed by a slower uptake of haem, which was inhibited by a large excess of unlabelled haemin-BSA, but not by BSA. However, accumulated haemin was not chased out of the vesicles by unlabelled haemin-BSA, suggesting specific binding of haemin with the membrane or transport into the lumen of the vesicle. Neither ATP nor a protonmotive force (delta(p)) generated by lactate oxidation was required for haemin binding or subsequent transport, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), sodium vanadate and monensin had no effect on haemin transport. The rate of haemin uptake following the initial rapid binding was proportional to the external haemin concentration, suggesting that the uptake process was driven by the haemin concentration gradient across the cell membrane. The kinetics of [14C]haemin uptake were similar in wild-type and cydD1 or delta(ccmA) mutants, suggesting that the activity of neither the CydDC nor CcmAB transporters is essential for haem export to the periplasm. Cytochrome d levels were unaffected by mutations in trxB (encoding thioredoxin reductase), trxA (thioredoxin), or grx (glutaredoxin), suggesting that the CydDC transporter does not export these components of reducing pathways for cytochrome assembly.

ATP-Binding Cassette Transporters↗
PubMed2000

Ubiquinone limits oxidative stress in Escherichia coli.

Ubiquinone is an essential redox component of the aerobic respiratory chains of bacteria and mitochondria. It is well established that mammalian ubiquinone can function in its reduced form (ubiquinol) as a lipid-soluble antioxidant preventing lipid peroxidation. The objective of this study was to test the hypothesis that prokaryotic ubiquinone is involved in the defence against oxidative stress in the cytoplasmic membrane. The rate of superoxide production by rapidly respiring wild-type Escherichia coli membranes was twofold higher than in the slowly respiring membranes from a ubiCA knockout mutant. However, large amounts of superoxide accumulated in the Ubi- membranes compared to wild-type membranes, which possess superoxide-scavenging ubiquinol. Likewise, the rate of H2O2 production was twofold higher in the wild-type, but the overall production of H2O2 was again significantly higher in the Ubi- membranes. Inclusion of a water-soluble ubiquinone homologue (UQ-1) effectively decreased the amount of H2O2 produced in the Ubi- membranes in a concentration-dependent manner. Addition of UQ-2 to the membranes was even more effective in limiting accumulation of H2O2 than was UQ-1, suggesting a role for the side-chain in conferring liposolubility in the antioxidative defence mechanism. Intracellular H2O2 concentration was increased 1.8-fold in the ubiCA mutant, and expression of the katG gene, encoding the catalase hydroperoxidase I, as well as catalase enzyme activity, were increased twofold in this mutant. The ubiCA mutant was hypersensitive to oxidative stress mediated by CuSO4 or H2O2; sensitivity to the latter could be abolished by addition of cysteine. This phenotype was also exhibited by a ubiG mutant, defective in the last step of UQ biosynthesis and therefore expected to accumulate several UQ biosynthetic intermediates. These observations support the participation of reduced ubiquinone as an antioxidant in E. coli. The ubiCA mutant exhibited a pleiotropic phenotype, being resistant to heat, linolenic acid and phleomycin. Resistance to the two latter compounds is probably due to reduced uptake. Like mutants unable to synthesize the quinol oxidase, cytochrome bd, the ubiCA mutant was also sensitive to dithiothreitol, an effect that is attributed to inability of the respiratory chain to maintain an appropriate redox balance in the periplasm.

Escherichia coli↗
PubMed2000

Cyanide inhibits respiration yet stimulates aerobic growth of Zymomonas mobilis.

Potassium cyanide at submillimolar concentrations (20-500 microM) inhibited the high respiration rates of aerobic cultures of Zymomonas mobilis but, remarkably, stimulated culture growth. In batch culture, after an extended lag phase, exponential growth persisted longer, resulting in higher biomass densities. In aerobic chemostat cultures, elevated biomass concentration was observed in the presence of cyanide. This growth stimulation effect is attributed to decreased production of the inhibitory metabolite acetaldehyde at lowered respiration rates, when more reducing equivalents are channelled to alcohol dehydrogenase. Growth in the presence of cyanide did not alter the membrane cytochrome content. In non-growing cyanide-preincubated cells, with ethanol as the respiratory substrate, cyanide increased ATP levels; in such cells, a large part of the cyanide-sensitive respiration was inhibited within a few seconds after ethanol addition, while inhibition of the rest of respiration took several minutes. The more cyanide-sensitive respiration was apparently energy-nongenerating, and was absent in membrane preparations. Pelleting of membranes from cell-free extracts produced 'soluble' fractions in which a b-type haem was detectable by reduced minus oxidized difference spectroscopy. The function of the Z. mobilis respiratory chain in cell growth and respiratory protection, and the possible physiological role of aerobic generation of inhibitory metabolites, are discussed.

Acetaldehyde↗
PubMed1999

An unusual cytochrome o'-type cytochrome c oxidase in a Bacillus cereus cytochrome a3 mutant has a very high affinity for oxygen.

Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spectrally detectable cytochromes aa3 or caa3. The nature of the remaining oxidase(s) catalysing oxygen uptake has been studied. Respiratory oxidase activities and the levels of cytochromes b and c increased 2.6- to 4.2-fold on transition from exponential growth, in either of two media, to sporulation stage III, as previously observed for the parent wild-type strain. NADH oxidase activity at both stages of culture was several-fold higher than ascorbate plus tetramethyl-p-phenylenediamine (TMPD) oxidase activity, consistent with the TMPD- phenotype of strain PYM1. Oxidase activity with ascorbate as substrate was significant even in the absence of TMPD as electron mediator, suggesting that the terminal oxidase receives electrons from a cytochrome c. Carbon monoxide (CO) difference spectra of membranes were obtained using various reductants (ascorbate +/- TMPD, NADH, dithionite) and revealed a haemoprotein resembling cytochrome o'. The CO complex of this cytochrome was photodissociable: the photodissociation spectrum (photolysed minus CO-ligated) exhibited a trough at 416 nm and a peak at 436 nm, together with minor features in the alpha/beta region of the spectrum, consistent with the presence of a cytochrome o'-like pigment. CO recombination occurred at -85 to -95 degrees C. No other haemoproteins showing photoreversible CO binding under these conditions were detected. Evidence that this pigment was the oxidase responsible for substrate oxidation was obtained by photodissociating the CO complex at subzero temperatures in the presence of oxygen; this resulted in faster ligand recombination, attributed to oxygen binding, and extensive oxidation of cytochromes c and b. The oxygen affinity of the oxidase was determined by using the deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissociated oxygen concentration. A single oxidase was revealed with a K(m) for oxygen of about 8 nM; this is one of the highest affinities yet reported for a terminal oxidase.

Bacillus cereus↗
PubMed1998

A factor produced by Escherichia coli K-12 inhibits the growth of E. coli mutants defective in the cytochrome bd quinol oxidase complex: enterochelin rediscovered.

Escherichia coli produces an extracellular factor that inhibits the aerobic growth of Cyd- mutants, defective in the synthesis or assembly of the cytochrome bd-type quinol oxidase. This paper shows that such a factor is the iron-chelating siderophore enterochelin. Mutants in entA or aroB, defective in the production of enterochelin, did not produce the factor that inhibits the growth of cydAB and cydDC mutants; purified enterochelin inhibited the growth of Cyd- mutants, but not that of wild-type cells. Other iron-chelating agents, particularly ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA), whose complex with Fe(III) has a large stability constant (log K = 33.9), also inhibited the growth of Cyd- mutants at micromolar concentrations, but not that of wild-type cells. Supplementation of agar plates with Fe(III) or boiled catalase prevented the inhibition of Cyd- mutants by the extracellular factor. Spontaneous mutants isolated by being able to grow in the presence of the extracellular factor on plates also showed increased resistance to iron chelators. The reducing agent ascorbate, ascorbate plus In(III), ascorbate plus Ga(III), or Ga(III) alone, also alleviated inhibition by the extracellular factor, presumably by reducing iron to Fe(II) and complexing of the siderophore with alternative trivalent metal cations. The preferential inhibition of Cyd- mutants by the extracellular factor and other iron chelators is not due to decrease in expression, activity or assembly of cytochrome bo', the major alternative oxidase mediating quinol oxidation. Cyd- mutants overproduce siderophores, presumably reflecting intracellular iron deprivation.

Ascorbic Acid↗
PubMed1998

Requirement for ubiquinone downstream of cytochrome(s) b in the oxygen-terminated respiratory chains of Escherichia coli K-12 revealed using a null mutant allele of ubiCA.

An Escherichia coli knockout ubiCA mutant has been constructed using a gene replacement method and verified using both Southern hybridization and PCR. The mutant, which was unable to synthesize ubiquinone (Q), showed severely diminished growth yields aerobically but not anaerobically with either nitrate or fumarate as terminal electron acceptors. Low oxygen uptake rates were demonstrated in membrane preparations using either NADH or lactate as substrates. However, these rates were greatly stimulated by the addition of ubiquinone-1 (Q-1). The rate of electron transfer to those oxidase components observable by photodissociation of their CO complexes was studied at sub-zero temperatures. In the ubiCA mutant, the reduced form of haemoproteins--predominantly cytochrome b595--was reoxidized significantly faster in the presence of oxygen than in a Ubi+ strain, indicating the absence of Q as electron donor. Continuous multiple-wavelength recordings of the oxidoreduction state of cytochrome(s) b during steady-state respiration showed greater reduction in membranes from the ubiCA mutant than in wildtype membranes. A scheme for the respiratory electron-transfer chain in E. coli is proposed, in which Q functions downstream of cytochrome(s) b.

Aerobiosis↗
PubMed1998

Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry.

A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b595 and b560. The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx. 5 microM). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.

Azotobacter vinelandii↗