Redox-linked protonation of cytochrome c oxidase: the effect of chloride bound to CuB

The effect of bound Cl- on the redox-linked protonation of soluble beef heart cytochrome c oxidase (CcOX) has been investigated at pH 7.3-7.5 by multiwavelength stopped-flow spectroscopy, using phenol red as the pH indicator in an unbuffered medium. Reduction by Ru-II hexamine of the Cl-bound enzyme is associated with an overall apparent uptake of 1.40 +/- 0.21 H+/aa3, whereas 2.28 +/- 0.36 H+/aa3 is taken upon reduction of the Cl-free enzyme. Bound Cl- has no effect on the extent of H+ uptake coupled to heme a reduction (0.59 +/- 0.06 H+/aa3), but significantly decreases (by approximately 0.9 H+/aa3) the apparent stoichiometry of H+ uptake coupled to heme a3-Cu(B) reduction, by eliminating the net H+ uptake linked to Cu(B) reduction. To account for these results, we propose that, after the transfer of the first electron to the active site, reduction of Cu(B) is associated with Cl- dissociation, addition of a H+, and diffusion into the bulk (with subsequent dissociation) of HCl. In the physiologically competent Cl--free enzyme, an OH- likely bound to oxidized Cu(B) is protonated upon arrival of the first electron, and dissociates as H2O. The relevance of this finding to the understanding of the enzyme mechanism is discussed.     

Steady-state H+/O stoichiometry of liver mitochondria.

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.     

Iron oxidation chemistry in ferritin. Increasing Fe/O2 stoichiometry during core formation

The origin of previously observed variations in stoichiometry of iron oxidation during the oxidative deposition of iron in ferritin has been poorly understood. Knowledge of the stoichiometry of Fe(II) oxidation by O2 is essential to establishing the mechanism of iron core formation. In the present work, the amount of Fe(II) oxidized was measured by M?ssbauer spectrometry and the O2 consumed by mass spectrometry. The number of protons produced in the reaction was measured by "pH stat" titration and hydrogen peroxide production by the effect of the enzyme catalase on the measured stoichiometry. For protein samples containing low levels of iron (24 Fe(II)/protein) the stoichiometry was found to be 1.95 +/- 0.18 Fe(II)/O2 with H2O2 being a product, viz. Equation 1. 2Fe2+ + O2 + 4H2O----2FeOOH + H2O2 + 4H+ (1) EPR spin trapping experiments showed no evidence of superoxide radical formation. The stoichiometry markedly increased with additional iron (240-960 Fe/protein), to a value of 4 Fe(II)/O2 as in Equation 2. 4Fe2+ + O2 + 6H2O----4FeOOH + 8H+ (2) As the iron core is progressively laid down, the mechanism of iron oxidation changes from a protein dominated process with H2O2 being the primary product of O2 reduction to a mineral surface dominated process where H2O is the primary product. These results emphasize the importance of the apoferritin shell in facilitating iron oxidation in the early stage of iron deposition prior to significant development of the polynuclear iron core.     

H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli

A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.     

Stoichiometry of mitochondrial H+ translocation coupled to succinate oxidation at level flow

The mechanistic stoichiometry of vectorial H+ translocation coupled to succinate oxidation by rat liver mitochondria in the presence of a permeant cation has been determined under level flow conditions with a membraneless fast responding O2 electrode kinetically matched with a glass pH electrode. The reactions were initiated by rapid injection of O2 into the anaerobically preincubated test system under conditions in which interfering H+ backflow was minimized. The rates of O2 uptake and H+ ejection, obtained from computer-fitted regression lines, were monotonic and first order over 75% of the course of O2 consumption. Extrapolation of the observed rates to zero time, at which zero delta mu H+ and thus level flow prevails, yielded vectorial H+/O flow ratios above 7 and closely approaching 8. The mitochondria undergo no irreversible change and give identical H+/O ratios on repeated tests. In a further refinement, the lower and upper limits of the mechanistic H+/O ratio were determined to be 7.55 and 8.56, respectively, from plots of the rates of O2 uptake versus H+ ejection at increasing malonate and increasing valinomycin concentrations, respectively. It is therefore concluded that the mechanistic H+/O ratio for energy-conserving sites 2 + 3 is 8, in confirmation of earlier measurements. KCl concentration is critical for maximal observed H+/O ratios. Optimum conditions and possible errors in determination of mechanistic H+/O translocation ratios are discussed.     

Harpin modulates the accumulation of salicylic acid by Arabidopsis cells via apoplastic alkalization

It is reported here that salicylic acid (SA) is rapidly taken up by Arabidopsis cells, and its uptake is accompanied by media alkalization and cytosolic acidification, and it is inhibited by the ionophore nigericin, suggesting that its import is linked with that of H+ and driven by a proton gradient. Such import and accumulation declined sharply within a narrow physiological pH range (pH 5.7-6.1), corresponding to a reduction in the [H+] of the media from 1.99 micromol l(-1) to 0.79 micromol l(-1). Following the initial uptake, SA was exported back into the media as free SA against a continued [H+]-dependent import. Since the uptake and accumulation of SA declines sharply within a narrow pH range and cell wall alkalization is an early response during incompatible plant/pathogen interactions, the bacterial elicitor harpin(Pss) was used to investigate how SA transport may be modulated during defence responses. Harpin induced a rapid and sustained alkalization of the cell suspension media, reaching the critical pH (pH 5.9-6.1) at which SA import is inhibited at c. 60 min. Such media alkalization corresponded with a reduction in the SA associated with cells co-treated with harpin, and an inhibition of SA uptake in cells pretreated with harpin. Scavengers of ROS, or compounds which generate H2O2 or NO had little effect on the import or net export of SA, suggesting that media alkalization induced by harpin is sufficient to modulate the kinetics of SA transport.     

Measurement of the proton-motive stoichiometry of the respiratory chain of rat liver mitochondria: the effect of N-ethylmaleimide

The apparent proton-motive stoichiometry as measured by the oxygen-pulse technique in KCl medium is depressed by the rapid uptake of inorganic phosphate, unless endogenous phosphate is depleted or uptake is inhibited. In sucrose or choline chloride media, where the internal pH is more acid than in KCl media, uptake may be greatly diminished. In the absence of significant phosphate uptake, the observed stoichiometry of around 8, obtained with no added substrate or respiratory inhibitors, appears to be characteristic of NADH oxidation without significant participation of the proton-translocating NAD(P) transhydrogenase. A mechanistic stoichiometry of at least 8 is indicated.     

Membrane-potential-dependent changes in the stoichiometry of charge translocation by the mitochondrial electron transport chain

The charge/oxygen (q+/O) stoichiometry of mitochondria respiring on succinate was measured under conditions of high membrane potential (delta psi). The technique used was a variation of the steady-state method of Al-Shawi and Brand [(1981) Biochem. J. 200, 539-546]. We show that q+/O was about 2.7 at high values of delta psi (170 mV). As delta psi was lowered from 170 mV to 85 mV with the respiratory inhibitor malonate the q+/O stoichiometry increased to 6.0. A number of artefacts which could have led to an underestimation of the q+/O stoichiometry were eliminated. These included effects of any rapid change in mitochondrial volume, internal pH, activity of the endogenous K+/H+ exchanger or in H+ conductance due to changes in delta psi after the addition of inhibitor. The experiments presented here are the first direct demonstration that the stoichiometry of proton pumping by the mitochondrial respiratory chain changes as delta psi is varied.     

Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.     

Sodium-dependent phosphate transport by apical membrane vesicles from a cultured renal epithelial cell line (LLC-PK1)

Apical membrane vesicles were prepared from confluent monolayers of LLC-PK1 cells grown upon microcarrier beads. The final membrane preparation, obtained by a modified divalent cation precipitation technique, was enriched in alkaline phosphatase, leucine aminopeptidase and trehalase (8-fold compared to the initial homogenate). Analysis of phosphate uptake into the vesicles identified a specific sodium-dependent pathway. Lithium and other cations were unable to replace sodium. At 100 mmol/l sodium and pH 7.4, an apparent Km for phosphate of 99 +/- 19 mumol/l and an apparent Ki for arsenate of 1.9 mmol/l were found. Analysis of the sodium activation of phosphate uptake gave an apparent Km for sodium of 32 +/- 12 mmol/l and suggested the involvement of two sodium ions in the transport mechanism. Sodium modified the apparent Km of the transport system for phosphate. The rate of sodium-dependent phosphate uptake was higher at pH 6.4 than at pH 7.4. At both pH values, an inside negative membrane potential (potassium gradient plus valinomycin) had no stimulatory effect on the rate of the sodium-dependent component of phosphate uptake. It is concluded that the apical membrane of LLC-PK1 cells contains a sodium-phosphate cotransport system with a stoichiometry of 2 sodium ions: 1 phosphate anion.     

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Upper and lower limits of the charge translocation stoichiometry of cytochrome c oxidase

The mechanistic stoichiometry of charge separation coupled to the flow of electrons through cytochrome c oxidase has remained a center of controversy since it was first demonstrated that cytochrome oxidase is an H+ pump. Currently the major dispute is whether the q+/O ratio for this segment is 4 or 6. One cause of the controversy is incomplete coupling between electron flow, electrogenic H+ ejection, and electrophoretic cation uptake, which is usually attributed to finite rates of H+ leakage and/or slippage of the H+ pumps. To minimize the uncertainty which incomplete coupling introduces into estimates of the mechanistic stoichiometry, a new approach (Beavis, A. D., and Lehninger, A. L. (1986) Eur. J. Biochem. 158, 307-314) has been used to determine the upper and lower limits of the mechanistic q+/O translocation stoichiometry of cytochrome oxidase. In this approach, the relationship between the rate of valinomycin-dependent K+ uptake, JK, and rate of O2 consumption, JO, is determined as the rates are modulated by two distinct means. When the rates are modulated by the rate of electron flow (i.e. rate of energy supply) the slope of JK versus JO must at all points be less than the mechanistic K+/O ratio. On the other hand, when the rates are modulated by varying the concentration of valinomycin (i.e. the rate of energy utilization) the slope of JK versus JO must at all points be greater than the mechanistic K+/O ratio. The results indicate that the q+/O ratio lies between 4.3 and 5.5. These data are inconsistent with both currently favored stoichiometries, and it is suggested that the true mechanistic stoichiometry of charge separation coupled to electron flow through cytochrome oxidase may be 5 q+/O.     

Stoichiometry of the topa quinone biogenesis reaction in copper amine oxidases

The stoichiometry of the topa quinone biogenesis reaction in phenylethylamine oxidase from Arthrobacter globiformis (AGAO) has been determined. We have shown that the 6e- oxidation of tyrosine to topa quinone (TPQ) consumes 2 mol of O2 and produces 1 mol of H2O2/mol of TPQ formed. The rate of H2O2 production is first-order (kobs = 1.0 +/- 0.2 min-1), a rate only slightly lower than the rate of TPQ formation directly determined previously (kobs = 1.5 +/- 0.2 min-1). This gives the following net reaction stoichiometry for TPQ biogenesis: E-Tyr + 2O2 --> E-TPQ + H2O2. This stoichiometry is in agreement with recently proposed mechanisms for TPQ biogenesis, and rules out several possible alternatives.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

The gastric [H,K]ATPase:H+/ATP stoichiometry

An H+/ATP ratio of 2 for H+ transport was determined from initial rate measurements at pH 6.1 in a purified gastric microsomal fraction containing the [H,K]ATPase. This ratio was independent of external KCl, though the apparent K0.5 for ATP was increased from 10.78 +/- 0.51 (n = 3) to 64.6 +/- 11.9 (n = 3) microM ATP and from 5.13 +/- 0.64 (n = 3) to 65.2 +/- 0.64 (n = 3) microM ATP for H+ transport and the K+-stimulated ATPase, respectively, as K+external was increased from 12 to 150 mM. The H+/ATP ratio was also relatively independent of ATP concentration. Maximum initial rates obtained in KCl-equilibrated vesicles were independent of added valinomycin, though net H+ transport was increased 29.3 +/- 1.03% (n = 6) by the addition of ionophore. Maximum net H+ transport in this vesicle preparation was 185 +/- 2.1 (n = 14) nmol mg-1 of protein. Initial rate measurements of ATPase represent a burst of K+-dependent activity of approximately 10-15 s duration. The H+/ATP stoichiometry was calculated based on the K+-stimulated component of hydrolysis. Under most conditions, the Mg2+-dependent component of hydrolysis was less than 10% of the (Mg2+ + K+) component of hydrolysis.     

Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Thrombin-induced platelet aggregation is affected by external Na+ independently of the Na+/H+ exchange

Thrombin affects blood platelets by activation of Na+/H+ exchange and induction of aggregation, but the relationship between these effects is under debate. The present study attempts to clarify whether the activation of the exchanger activity is required for platelet aggregation. In apparent support of such a requirement, thrombin-induced aggregation is higher in Na+ medium than in N-methylglucamine+ medium and is inhibited by sphingosine, an inhibitor of protein kinase C known to regulate the Na+/H+ exchanger. However, the inhibition of aggregation by sphingosine occurs in both Na+-containing and Na+-free media, the aggregation is identical in Na+ and K+-containing media, and is not inhibited by 5-N-(3-aminophenyl)amiloride, at a concentration 10-fold higher than its Ki for platelet Na+/H+ exchange. Furthermore, at low concentration (0.005 U/ml) thrombin induces aggregation but does not activate the exchange. It is concluded that the activation of Na+/H+ exchange is not required for thrombin-induced platelet aggregation and that the apparent augmentation of aggregation by Na+ is due to an inhibitory effect of N-methylglucamine+.     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.