Aer on the inside looking out: paradigm for a PAS-HAMP role in sensing oxygen, redox and energy

Aer, the Escherichia coli aerotaxis (oxygen-sensing) receptor, is representative of a small class of receptors that face the cytoplasm in bacteria. Instead of sensing oxygen directly, Aer detects redox changes in the electron transport system or cytoplasm when the bacteria enter or leave a hypoxic microniche. As a result, Aer sensing also enables bacteria to avoid environments where carbon deficiency, unfavourable reduction potential or other insults would limit energy production. An FAD-binding PAS domain is the sensor for Aer and a HAMP domain interacts with the PAS domain to form an input-output module for signal transduction. By analogy to the first solution structure of an isolated HAMP domain from Archaeoglobus, Aer HAMP is proposed to fold into a four-helix bundle that rotates between a signal-on and signal-off conformation. Aer is the first protein in which a PAS-HAMP input-output module has been investigated. The structure and signal transduction mechanism of Aer is providing important insights into signalling by PAS and HAMP domains.     

In search of higher energy: metabolism-dependent behaviour in bacteria

Bacteria use different strategies to navigate to niches where environmental factors are favourable for growth. Chemotaxis is a behavioural response mediated by specific receptors that sense the concentration of chemicals in the environment. Recently, a new type of sensor has been described in Escherichia coli that responds to changes in cellular energy (redox) levels. This sensor, Aer, guides the bacteria to environments that support maximal energy levels in the cells. A variety of stimuli, such as oxygen, alternative electron acceptors, light, redox carriers that interact with the electron transport system and metabolized carbon sources, effect changes in the cellular energy (redox) levels. These changes are detected by Aer and by the serine chemotaxis receptor Tsr and are transduced into signals that elicit appropriate behavioural responses. Diverse environmental signals from Aer and chemotaxis receptors converge and integrate at the level of the CheA histidine kinase. Energy sensing is widespread in bacteria, and it is now evident that a variety of signal transduction strategies are used for the metabolism-dependent behaviours. The occurrence of putative energy-sensing domains in proteins from cells ranging from Archaea to humans indicates the importance of this function for all living systems.     

Minimal requirements for oxygen sensing by the aerotaxis receptor Aer

The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared.     

Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa

It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion-insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.     

Differential activation of Escherichia coli chemoreceptors by blue-light stimuli

Enteric bacteria tumble, swim slowly, and are then paralyzed upon exposure to 390- to 530-nm light. Here, we analyze this complex response in Escherichia coli using standard fluorescence microscope optics for excitation at 440 +/- 5 nm. The slow swimming and paralysis occurred only in dye-containing growth media or buffers. Excitation elicited complete paralysis within a second in 1 muM proflavine dye, implying specific motor damage, but prolonged tumbling in buffer alone. The tumbling half-response times were subsecond for onset but more than a minute for recovery. The response required the chemotaxis signal protein CheY and receptor-dependent activation of its kinase CheA. The study of deletion mutants revealed a specific requirement for either the aerotaxis receptor Aer or the chemoreceptor Tar but not the Tar homolog Tsr. The action spectrum of the wild-type response was consistent with a flavin, but the chromophores remain to be identified. The motile response processed via Aer was sustained, with recovery to either step-up or -down taking more than a minute. The response processed via Tar was transient, recovering on second time scales comparable to chemotactic responses. The response duration and amplitude were dependent on relative expression of Aer, Tar, and Tsr. The main response features were reproduced when each receptor was expressed singly from a plasmid in a receptorless host strain. However, time-resolved motion analysis revealed subtle kinetic differences that reflect the role of receptor cluster interactions in kinase activation-deactivation dynamics.     

Rhein as an electron acceptor for various flavoproteins and for electron transport particles

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) which has been previously employed as an inhibitor for electron transport particles, NADH dehydrogenase, and other flavoproteins is reducible under physiological conditions. Soluble hydrogenase from Alcaligenes eutrophus H 16, several flavoproteins, and electron transport particles from baker's yeast and from beef heart were found to catalyse NADH oxidation with 9 micrometers to 2mM rhein as the electron acceptor. Dithionite or enzymatically reduced rhein (lambda max = 408 nm) is immediately reoxidized to rhein lambda max = 437 nm) by oxygen. Cyclovoltagrams reveal the midpoint redox potentials --0.240 V, -0.270 V, -0.280 V, -0.335 V at pH 6.0, 7.0, 7.7, 9.2, respectively. Due to its redox behaviour, caution should be exercised using rhein as a flavin-site-directed inhibitor for biological electron transfer systems.     

Inversion of aerotactic response in Escherichia coli deficient in cheB protein methylesterase.

Mutants of Escherichia coli and Salmonella typhimurium that were deficient in protein methylesterase activity encoded by cheB had an inverted response to oxygen; they were repelled by concentrations of oxygen that attract wild-type bacteria. Normal responses to oxygen and phosphotransferase substrates were observed in mutants that were deficient in protein methyltransferase (CheR) and the methyl-accepting transducing proteins (Tsr, Tar, Trg). However, the methylation-independent response to oxygen was modified by the loss of esterase activity. The inversion was apparently effected by the amidated Tsr protein present in cheB tsr+ mutants because aerotaxis was normal in cheB tsr strains. Chemotaxis to phosphotransferase sugars was normal in cheB mutants provided the extreme clockwise bias of the flagellar motors was modified to increase the probability of counterclockwise rotation.     

H+/e- stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells.

Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H+/e- stoichiometry being close to 2.5 and 1.5, respectively. In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O2 and to 0 for DMSO. These data suggest that (i) the H+/e- stoichiometry for E. coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.     

Energy taxis is the dominant behavior in Azospirillum brasilense

Energy taxis encompasses aerotaxis, phototaxis, redox taxis, taxis to alternative electron acceptors, and chemotaxis to oxidizable substrates. The signal for this type of behavior is originated within the electron transport system. Energy taxis was demonstrated, as a part of an overall behavior, in several microbial species, but it did not appear as the dominant determinant in any of them. In this study, we show that most behavioral responses proceed through this mechanism in the alpha-proteobacterium Azospirillum brasilense. First, chemotaxis to most chemoeffectors typical of the azospirilla habitat was found to be metabolism dependent and required a functional electron transport system. Second, other energy-related responses, such as aerotaxis, redox taxis, and taxis to alternative electron acceptors, were found in A. brasilense. Finally, a mutant lacking a cytochrome c oxidase of the cbb(3) type was affected in chemotaxis, redox taxis, and aerotaxis. Altogether, the results indicate that behavioral responses to most stimuli in A. brasilense are triggered by changes in the electron transport system.     

Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid-helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe-1 (oxidised), 1e-1 (semiquinone), 2e-1 (mostly semiquinone and reduced Fe2S2), and 3e-1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd-electron forms is consistent with its proposed role as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for passage to component A in one-electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.) The mid-point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD., Em = -150 mV; Fe2S2/Fe2.S2,Em = -220 mV; FAD./FAD..,Em = -260 mV. the presence of NADH did not alter these mid-point potentials. These mid-point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em = -320 mV) onto component A (Em = +150 mV and Em = -150 mV). The reducing power from NADH appears to be required by component A to activate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

Signal transduction in chemotaxis to oxygen in Escherichia coli and Salmonella typhimurium.

Pathways previously proposed for sensory transduction in chemotaxis to oxygen (aerotaxis) involved either (i) cytochrome o, the electron transport system, and proton motive force or (ii) enzyme IIGlucose and the phosphoenolpyruvate:carbohydrate phosphotransferase system for active transport. This investigation distinguished between these possibilities. Aerotaxis was absent in a cyo cyd strain of Escherichia coli that lacked both cytochrome o and cytochrome d, which are the terminal oxidases for the branched electron transport system in E. coli. Aerotaxis, measured by either a spatial or temporal assay, was normal in E. coli strains that had a cyo+ or cyd+ gene or both. The membrane potential of all oxidase-positive strains was approximately -170 mV in aerated medium at pH 7.5. Behavioral responses to changes in oxygen concentration correlated with changes in proton motive force. Aerotaxis was normal in ptsG and ptsI strains that lack enzyme IIGlucose and enzyme I, respectively, and are deficient in the phosphotransferase system. A cya strain that is deficient in adenylate cyclase also had normal aerotaxis. We concluded that aerotaxis was mediated by the electron transport system and that either the cytochrome d or the cytochrome o branch of the pathway could mediate aerotaxis.     

Aerotaxis in Salmonella typhimurium: role of electron transport

Sensory transduction in aerotaxis required electron transport, in contrast to chemotaxis, which is independent of electron transport. Assays for aerotaxis were developed by employing spatial and temporal oxygen gradients imposed independently of respiration. By varying the step increase in oxygen concentration in the temporal assay, the dose-response relationship was obtained for aerotaxis in Salmonella typhimurium. A half-maximal response at 0.4 microM oxygen and inhibition by 5 mM KCN suggested that the "receptor" for aerotaxis is cytochrome o. The response was independent of adenosine triphosphate formation via oxidative phosphorylation but did correlate with changes in membrane potential monitored with the fluorescent cyanine dye diS-C3-(5). Nitrate and fumarate, which are alternative electron acceptors for the respiratory chain in S. typhimurium, inhibited aerotaxis when nitrate reductase and fumarate reductase were induced. These results support the hypothesis that taxis to oxygen, nitrate, and fumarate is mediated by the electron transport system and by changes in the proton motive force. Aerotaxis was normal in Escherichia coli mutants that were defective in the tsr, tar, or trg genes; in S. typhimurium, oxygen did not stimulate methylation of the products of these genes. A cheC mutant which shows an inverse response to chemoattractants also gave an inverse response to oxygen. Therefore, aerotaxis is transduced by a distinct and unidentified signally protein but is focused into the common chemosensory pathway before the step involving the cheC product. When S. typhimurium became anaerobic, the decreased proton motive force from glycolysis supported slow swimming but not tumbling, indicating that a minimum proton motive force was required for tumbling. The bacteria rapidly adapted to the anaerobic condition and resumed tumbling after about 3 min. The adaptation period was much shorter when the bacteria had been previously grown anaerobically.     

Function of the N-terminal cap of the PAS domain in signaling by the aerotaxis receptor Aer

Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers.     

Methylation-independent aerotaxis mediated by the Escherichia coli Aer protein

Aer is a membrane-associated protein that mediates aerotactic responses in Escherichia coli. Its C-terminal half closely resembles the signaling domains of methyl-accepting chemotaxis proteins (MCPs), which undergo reversible methylation at specific glutamic acid residues to adapt their signaling outputs to homogeneous chemical environments. MCP-mediated behaviors are dependent on two specific enzymes, CheR (methyltransferase) and CheB (methylesterase). The Aer signaling domain contains unorthodox methylation sites that do not conform to the consensus motif for CheR or CheB substrates, suggesting that Aer, unlike conventional MCPs, might be a methylation-independent transducer. Several lines of evidence supported this possibility. (i) The Aer protein was not detectably modified by either CheR or CheB. (ii) Amino acid replacements at the putative Aer methylation sites generally had no deleterious effect on Aer function. (iii) Aer promoted aerotactic migrations on semisolid media in strains that lacked all four of the E. coli MCPs. CheR and CheB function had no influence on the rate of aerotactic movements in those strains. Thus, Aer senses and signals efficiently in the absence of deamidation or methylation, methylation changes, methylation enzymes, and methyl-accepting chemotaxis proteins. We also found that chimeric transducers containing the PAS-HAMP sensing domain of Aer joined to the signaling domain and methylation sites of Tar, an orthodox MCP, exhibited both methylation-dependent and methylation-independent aerotactic behavior. The hybrid Aear transducers demonstrate that methylation independence does not emanate from the Aer signaling domain but rather may be due to transience of the cellular redox changes that are thought to trigger Aer-mediated behavioral responses.     

Dissipation of Proton Motive Force is not Sufficient to Induce the Phage Shock Protein Response in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H⁺-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response.     

Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer

The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 alpha-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer.     

A multifunctional hydroquinone oxidase of the external cell surface and sera.

A multifunctional cell surface protein with NADH oxidase (NOX) activity and capable of oxidizing hydroquinones is located at the exterior of the cell and is shed in soluble form into sera. The oxidase appears to function as a terminal oxidase of a trans plasma membrane electron transport chain consisting of a NAD(P)H-ubiquinone reductase at the cytosolic membrane surface, possibly a b-type cytochrome, ubiquinone and the oxidase. Hyperactivity or conditions that interrupt ordered 2H+ + 2e- transport from NAD(P)H or hydroquinone to molecular oxygen and other acceptors at the external cell surface may result in the generation of superoxide. The latter may serve to propagate aging-related redox changes both to adjacent cells and circulating blood components. A circulating NOX activity form associated with aging and the reduction of cytochrome c by sera of aged patients that is partially inhibited by ubiquinone are described.     

PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding

The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fold.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.