A non-equilibrium thermodynamics analysis of active transport within the framework of the chemiosmotic theory

The proton circuit devised by Mitchell in the chemiosmotic theory was subjected to analysis using the formalism of irreversible thermodynamics. The phenomenological coefficients and the degree of coupling relating co-permeant flows were derived for anion/H⁺, substrate/H⁺, cation/H⁺ and anion/anion biporter models. Linearity and equality of the cross-coefficients in Onsager relations were always satisfied. Macroscopic flows leading to charges splitting, such as oxido-reduction, hydro-dehydratation and transhydrogenase, are driven by a composite thermodynamic force which includes the proton-motive component. Multiple coupling occurs in the circuit when it is assumed that the net inward flux of protons becomes zero, i.e. when the circulation of protons reaches a stationary state. Under these conditions, oxidative phosphorylation, ATPase- or respiration-linked transhydrogenase and uptake of anion or cation against their electrochemical gradient may be predicted, in agreement with known experimental evidence.     

Colonic absorption of inorganic ions and volatile fatty acids in the rabbit

Net fluxes of water, Na+, K+, Cl-, HCO3- and volatile fatty acids (VFA) were investigated in three different segments of rabbit colon. Two opposite phenomena occurred: secretion of water and inorganic ions in the oral part of the colon and absorption in the remaining colon; VFA were always absorbed. The movement of cations was closely correlated with those of VFA and Cl-. Results are consistent with the presence of exchange: Na+/H+, K+/H+, in the colon brush border membrane. In fact net absorption of cations and VFA seems linked to the availability of protons. In the absence of net cation transport an additional source of protons may be provided by hydration of luminal CO2. So VFA could enter mucosa by passive diffusion as the undissociated acids.     

The broadly selective human Na+/nucleoside cotransporter (hCNT3) exhibits novel cation-coupled nucleoside transport characteristics

The concentrative nucleoside transporter (CNT) protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. hCNT3, a Na+/nucleoside symporter, transports a broad range of physiological purine and pyrimidine nucleosides as well as anticancer and antiviral nucleoside drugs, and belongs to a different CNT subfamily than hCNT1/2. H+-dependent Escherichia coli NupC and Candida albicans CaCNT are also CNT family members. The present study utilized heterologous expression in Xenopus oocytes to investigate the specificity, mechanism, energetics, and structural basis of hCNT3 cation coupling. hCNT3 exhibited uniquely broad cation interactions with Na+, H+, and Li+ not shared by Na+-coupled hCNT1/2 or H+-coupled NupC/CaCNT. Na+ and H+ activated hCNT3 through mechanisms to increase nucleoside apparent binding affinity. Direct and indirect methods demonstrated cation/nucleoside coupling stoichiometries of 2:1 in the presence of Na+ and both Na+ plus H+, but only 1:1 in the presence of H+ alone, suggesting that hCNT3 possesses two Na+-binding sites, only one of which is shared by H+. The H+-coupled hCNT3 did not transport guanosine or 3'-azido-3'-deoxythymidine and 2',3'-dideoxycytidine, demonstrating that Na+- and H+-bound versions of hCNT3 have significantly different conformations of the nucleoside binding pocket and/or translocation channel. Chimeric studies between hCNT1 and hCNT3 located hCNT3-specific cation interactions to the C-terminal half of hCNT3, setting the stage for site-directed mutagenesis experiments to identify the residues involved.     

Protons as substitutes for sodium and potassium in the sodium pump reaction

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.     

Diphtheria toxin-induced channels in Vero cells selective for monovalent cations

Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of 45Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed.     

Insights into the biochemistry of the ubiquitous NhaP family of cation/H+ antiporters

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Models for biological ion exchangers. I. Proton magnetic resonance studies of water structure in Bio-Rex 70.

Bio-Rex 70, a carboxylic acid cation exchanger, is studied as a biological ion-exchanger resin model for cellular cytoplasm. High-resolution proton magnetic resonance spectra of 1l ionic forms of Bio-Rex 70 are determined. From measured cation exchange capacities, water contents, and chemical shifts for the resin-phase water protons, the dependence of the chemical shift on the counter ion is calculated. The observed chemical shifts (Hz/geq/kg internal water, referred to the Ba2+ form) for each ionic form are: H+, --0.5; Li+, --0.2; Na+, 1.2; K+, 2; Rb+, 2.1; Ag+, --0.4; NH4+, --2.0; NMe4+, 1.2; NEt4+, 1.8; Mg2+, --2.0; Ca2+, --1.5; Sr2+, --0.6; Ba2+, 0.0 Zn2+, --2.3; Cd2+, --4.7; and La3+, --3.3. The results are in good agreement with earlier studies on Dowex 50, indicating that the carboxylate ion exchanger behaves like a concentrated polyelectrolyte. The widths at half-height for the internal water peaks of the polyvalent forms are quite large, ranging from 40 to 100 Hz.     

Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to potassium by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; it thus appeared that ACh leakage occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyse monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP), a rapid but transient ACh release was observed. Surprisingly, nigericin which also catalyses K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 promoted ACh release only when calcium (and not magnesium) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this calcium-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.     

Proton transport catalyzed by the sodium pump. Ouabain-sensitive ATPase activity and the phosphorylation of Na,K-ATPase in the absence of sodium ions

The possibility that H+ might substitute for Na+ at Na+ sites of Na+,K+-ATPase was studied. Na+,K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+,K+-ATPase). The specific activity was 1.1 mumol Pi/mg/min at pH 5.7, whereas the specific activity of Na+,K+-ATPase was 14 mumol Pi/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+,K+-ATPase was inhibited as effectively as Na+,K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+,K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.     

Sodium-coupled ATP synthesis in the bacterium Vitreoscilla.

The bacterium Vitreoscilla generates an electrical potential gradient due to sodium ion (delta psi Na+) across its membrane via respiratory-driven primary Na+ pump(s). The role of the delta psi Na+ as a driving force for ATP synthesis was, therefore, investigated. In respiring starved cells pulsed with 100 mM external Na+ [( Na+]o) there was a 167% net increase in cellular ATP concentration over basal levels compared with 0, 56, 78, and 78% for no addition, choline, Li+, and K+ controls, respectively. Doubling the [Na+]o to 200 mM boosted the net increase to 244% but a similar doubling of the choline caused only an increase to 78%. When the initial condition was intracellular Na+ ([Na+]i) = [Na+]o = 100 mM, there was a 94% net increase in cellular ATP compared with only 18 and 11% for Li+ and K+ controls, respectively, indicating that Nai+ may be the only cation tested that the cells extruded to generate the electrochemical gradient required to drive ATP synthesis. The Na(+)-dependent ATP synthesis was inhibited completely by monensin (12 microM), but only transiently by the protonophore 3,5-di-tert-butyl-4-hydroxybenzaldehyde (100 microM), further evidence that the Na+ gradient and not a H+ gradient was driving the ATP synthesis. ATP synthesis in response to an artificially imposed H+ gradient (delta pH approximately 3) in the absence of an added cation, or in the presence of Li+, K+, or choline, yielded similar delta ATP/delta pH ratios of 0.98-1.22. In the presence of Na+, however, this ratio dropped to 0.23, indicating that Na+ inhibited H(+)-coupling to ATP synthesis and possibly that H+ and Na+ coupling to ATP synthesis share a common catalyst. The above evidence adds to previous findings that under normal growth conditions Na+ is probably the main coupling cation for ATP synthesis in Vitreoscilla.     

The proton ladder,a static mechanism for ion/proton coports and counterports

An ion/proton counterport is formed simply by locating a chain of ionizable residues connected by a proton conducting path near a passive ion pore which spans the membrane. The electric coupling between the ion in transit through the pore and the residues can ensure that for each ion passing through the pore in one direction a proton is driven along the chain of ionizable residues (the proton ladder) in the same or in the opposite direction. The mechanism is symmetrical in that a trans-membrane ion gradient may drive protons against their electrochemical potential gradient or a proton gradient may drive ions against theirs. The mechanism is applicable to cation or anion channels and to coports or counterports. No mechanical motion is required other than the motion of the ions and the protons. Monte Carlo computer simulations are performed on the model and its predicted properties are listed. The new type of counterport model is compared with currently used models. Offprint requests to: D. T Edmonds     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.     

Rhizobium meliloti fixGHI sequence predicts involvement of a specific cation pump in symbiotic nitrogen fixation.

We present genetic and structural analyses of a fix operon conserved among rhizobia, fixGHI from Rhizobium meliloti. The nucleotide sequence of the operon suggests it may contain a fourth gene, fixS. Adjacent open reading frames of this operon showed an overlap between TGA stop codons and ATG start codons in the form of an ATGA motif suggestive of translational coupling. All four predicted gene products contained probable transmembrane sequences. FixG contained two cysteine clusters typical of iron-sulfur centers and is predicted to be involved in a redox process. FixI was found to be homologous with P-type ATPases, particularly with K+ pumps from Escherichia coli and Streptococcus faecalis but also with eucaryotic Ca2+, Na+/K+, H+/K+, and H+ pumps, which implies that FixI is a pump of a specific cation involved in symbiotic nitrogen fixation. Since prototrophic growth of fixI mutants appeared to be unimpaired, the predicted FixI cation pump probably has a specifically symbiotic function. We suggest that the four proteins FixG, FixH, FixI, and FixS may participate in a membrane-bound complex coupling the FixI cation pump with a redox process catalyzed by FixG.     

Proton-coupled organic cation transport in renal brush-border membrane vesicles

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H+ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294-298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N1-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [3H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the Km and increases the Vmax for NMN transport.     

Transport of cations and anions across forestomach epithelia: conclusions from in vitro studies

Secretion of saliva as well as absorptive and secretory processes across forestomach epithelia ensures an optimal environment for microbial digestion in the forestomachs. Daily salivary secretion of sodium (Na+) exceeds the amount found in plasma by a factor of 2 to 3, while the secretion of bicarbonate (HCO3-) is 6 to 8 times higher than the amount of HCO3- in the total extracellular space. This implies a need for efficient absorptive mechanisms across forestomach epithelia to allow for an early recycling. While Na+ is absorbed from all forestomachs via Na+/H+ exchange and a non-selective cation channel that shows increased conductance at low concentrations of Mg2+, Ca2+ or H+ in the luminal microclima and at low intracellular Mg2+, HCO3- is secreted by the rumen for the buffering of ingesta but absorbed by the omasum to prevent liberation of CO2 in the abomasum. Fermentation provides short chain fatty acids and ammonia (NH3) that have to be absorbed both to meet nutrient requirements and maintain ruminal homeostasis of pH and osmolarity. The rumen is an important location for the absorption of essential minerals such as Mg2+ from the diet. Other ions can be absorbed, if delivered in sufficient amounts (Ca2+, Pi, K+, Cl- and NH4+). Although the presence of transport mechanisms for these electrolytes has been described earlier, our knowledge about their nature, regulation and crosstalk has increased greatly in the last years. New transport pathways have recently been added to our picture of epithelial transport across rumen and omasum, including an apical non-selective cation conductance, a basolateral anion conductance, an apical H+-ATPase, differently expressed anion exchangers and monocarboxylate transporters.     

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.     

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

Analysis of ion channels by modeling the osmotic effects of weak acids and bases

Summary This paper describes computer programs which may be used to identify and analyze cation and anion channels. Weak acids are used to increase intracellular proton concentrations and by so doing to promote the exchange of osmotically active cations with protons. The time course of cation exchange is readily identified from the changes in cell volume which accompany the net changes in osmotically active cations. Weak bases are used to identify and analyze hydroxyl/anion exchange by a comparable strategy. The model was able to produce data that agreed with experimental data in the literatur with an accuracy equal to experimental error. One program, called PROPIONATE, uses the weak acid, propionic acid, to identify cation channels such as the sodium-proton exchanger or the calcium-dependent, potassium channel. A second program, called BASE, is more general because either a weak acid such as propionic acid or a weak base such as ammonia may be used individually or together. When experimental data ara available, the programs may be used to calculate permeability coefficients for ion channels and the capacity of intracellular buffers. The programs may be used also in the design of experiments. Initial values may be assigned to intracellular and extracellular electrolyte and proton concentrations. Values for intracellular buffer capacity and channel permeabilities may be chosen. The program will then generate changes in ions, cell volume, and intracellular pH when either a weak acid, a weak base of combination of the two is added to the external medium.     

Comparison of the electron spin echo envelope modulation (ESEEM) for human lactoferrin and transferrin complexes of copper(II) and vanadyl ion

Copper(II) and vanadyl ions were bound to human milk lactoferrin or serum transferrin with carbonate or oxalate as the synergistic anion. Electron spin echo envelope modulation (ESEEM) due to nitrogen of a coordinated histidine imidazole was observed for both the copper and vanadyl complexes. For both metals, the modulation frequencies in the Fourier transforms of the data were similar for the two proteins and were weakly dependent on anion. When data in D2O/glycerol-d3 were compared with data in H2O/glycerol, the deep deuterium modulation indicated multiple exchangeable protons in the vicinity of the metals with at most one proton within about 2.9 A of the metal. The distribution of exchangeable protons around the metals as probed by ESEEM was the same, within experimental uncertainty, for the copper or vanadyl complexes with either carbonate or oxalate as the anion. When 13C-labeled oxalate was used as the synergistic anion, 13C-ESEEM was observed for both the copper and vanadyl complexes of lactoferrin and transferrin. The deeper 13C modulation for copper and vanadyl transferrin [13C]oxalate than for vanadyl transferrin [13C]carbonate suggests that both ends of the oxalate are bound to the metal in the transferrin and lactoferrin complexes.