Estimation of the distance between the divalent cation binding site of des-1-41-light chain-activated bovine plasma protein C and a nitroxide spin label attached to the active-site serine residue.

The paramagnetic effect of Mn2+ on the electron paramagnetic resonance spectrum of a nitroxide spin label covalently attached to the active-site serine residue of des-1-41-light chain bovine plasma-activated protein C, and situated at a distance of approximately 1.2 nm from this amino acid, has been utilized to estimate the distance on the enzyme surface between the single Mn2+ site and the free electron of the spin label. This distance has been found to be approx. 1.12 nm. A significant paramagnetic effect of Mn2+ on the spectrum of this same nitroxide spin label bound to activated protein C (APC) has been found. However, in this case distance calculations are complicated by the existence of a multiplicity of Mn2+ sites on APC. If it is assumed that a single Mn2+ site is responsible for the paramagnetic effect on the spectrum of the spin label, the interelectron distance on APC would be approx. 0.90 nm.     

Specific binding sites for cations in bacteriorhodopsin.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.     

A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase

The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.     

Study of lysozyme by a spin-label method in the 2-mm range

Two millimeter range ESR-spectroscopy was used to measure the values of magnetic resonance parameters of egg lysozyme samples modified with nitroxyl label 4-(iodine-acetamide)-2,2,6,6-tetramethylpiperidine-1-oxyl by hist-15 residue. It has been shown that in a lyophilic sample the spin label forms a hydrogen bond with the protein group and when the sample is moistened--with water molecules. Temperature changes of the pattern of ESR line are analysed. It is concluded that in the moistened samples within 230-320 K the nitroxyl fragment of the label gets engaged in anisotropic movement with preferable rotation around Z-axis of N-O. fragment with anisotropy coefficient 5 and mean correlation time tau congruent to 5 X 10(-7) divided by 5 X 10(-8) c.     

A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus--revealed by EPR spectroscopy

As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu2+. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu2+. The copper may be removed by extensive washing with EDTA. Titration with Ni2+ then induces spin-spin interactions with nitroxyl spin labels that are attached either to the unique Cys54, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni2+ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni2+-binding site on the protein is situated around 14-18 A from the spin label on Cys54, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.     

Interaction of a membrane preparation of Na+, K+-ATPase with a spin-labeled analog of ATP]

Interaction of membrane Na+, K+-ATPase preparation from brain gray matter with spin-labelled ATP analogue, in which free iminoxyl radical is joined as a result of 2'(3')-OH ribose groups acylation, is studied. The rotatory mobility of spin-labelled ATP analogue in Na+,K+-ATPase preparation is found to change in non-linear manner during temperature variation (the break-point on the curve being at 20-23degrees C). It correlates with temperature dependence of Na+,K+-ATPase and temperature dependence of lipid viscosity in the membranes, determined by means of hydrophobic spin probes. Substitution of Mg2+ ions with paramagnetic Mn2+ ions resulted in an intense magnetic dipole-dipole interaction between a spin label and Mn2+ ion, which indicated the formation of triple complex enzyme--spin-labelled ATP--Mn2+.     

Study of the effect of the microenvironment on magnetic resonance parameters of spin-labeled human serum albumin in a 2-mm ESR range

Basic values of g-tensor and Azz component of HF tensor of two spin labels and spin probe on HSA and nitroxyl radicals HO-15, HO-34 in the solvents of different polarity were measured by 2 mm band ESR of 2 mm range. Magnetic-resonance parameters of liophylized and water-solved spin-labeled HSA were shown to correspond to the parameters of the solvents of the label HO-15 and HO-34 in ethyl alcohol and water. A conclusion was drawn concerning the identity of microenvironment of the nitroxyl fragment of liophylized HSA and frozen solution of the label HO-15 and HO-34 in ethyl alcohol and solvatation of the nitroxyl fragment of spin-labeled HSA and label HO-15 (HO-34) by water molecules.     

Spin label study of slow molecular rotations of globular proteins using microwave saturation effects in electron paramagnetic resonance

Viscosity, temperature and ionic strength dependences of ESR microwave saturation parameters of spin labelled human oxyhemoglobin (Hb) and bovine serum albumin (BSA) have been studied. The piperidine and pyrrolidine nitroxyl derivatives of maleimide were used as covalent SH reagents for Hb and BSA and the same two derivatives of gamma-benzocarboline and spin labelled stearic acid were used as noncovalent spin probes for BSA. The effects of label binding tightness on ESR spectral parameters were considered. The rotational correlation times were determined using viscosity dependences of the separation of the outer hyperfine extrema and Stokes extrapolations at high viscosities. The ESR microwave saturation parameters of the spin labels were shown to depend just weakly on temperature (at constant eta/t) over the range 0-25 degrees and on g, A values but to be sensitive to protein rotational correlation times up to 10(-4) sec and also to the rotational anisotropy and to the relative motion of the spin label.     

The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components.

Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.     

Spin-labeling studies of urea-treated leucine aminopeptidase.

1. Leucine aminopeptidase (EC 3-4-11-1) from bovine eye lens was spin-labeled at the most reactive thiol groups with 2,2,6,6-tetramethyl-4-[2-iodoacetamido]-piperidine-1-oxyl. 2. Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B approximately 4:1 was estimated. In B a hydrophobic environment of label was observed. 3. Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B') with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B' shows a time-dependent increase of its extent at the expense of B and A' (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn2+. b) Mn2+ complexed with the protein is partly released producting hydrated Mn2+. c) After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B approximately 4:1 even after long incubation. 4. 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.     

Electron spin echo modulation and nuclear relaxation studies of staphylococcal nuclease and its metal-coordinating mutants

Electron spin echo envelope modulation (ESEEM) spectroscopy has been applied to the determination of the number of water molecules coordinated to the metal in the binary complex of staphylococcal nuclease with Mn2+, to the ternary enzyme-Mn2+-3',5'-pdTp complex, and to ternary complexes of a number of mutant enzymes in which metal-binding ligands have been individually altered. Quantitation of coordinated water is based on ESEEM spectral comparisons of Mn2+-EDTA and Mn2+-DTPA, which differ by a single inner sphere water, and with Mn2+-(H2O)6. It was found that Mn2+ in the ternary complex of the wild-type enzyme has a single additional coordinated water, as compared to Mn2+ in the binary complex, confirming earlier findings based on T1 measurements of bound water [Serpersu, E. H., Shortle, D. L., & Mildvan, A. S. (1987) Biochemistry 26, 1289-1300]. Ternary complexes of the mutant proteins D40E, D40G, and D21Y have the same number of water ligands as the ternary complex of the wild-type enzyme, while the D21E mutant has one less water ligand. In order to maintain octahedral coordination geometry, these findings require two additional ligands to Mn2+ from the protein in the binary complex of the wild-type enzyme, probably Glu 43 and Asp 19, and one additional ligand from the protein in the ternary D40G and D21E complexes. Other ESEEM studies of ternary Mn2+ complexes of wild-type, D21E, and D21Y mutants indicate the coordination by Mn2+ of a phosphate of 3',5'-pdTp, as demonstrated by a 31P contact interaction of 3.9 +/- 0.3 MHz. Magnetic interaction of Mn2+ with 31P could not be demonstrated with the D40G and D40E mutants, suggesting that metal-phosphate distances are greater in these mutants than in the wild-type protein. In a parallel NMR study of the paramagnetic effects of enzyme-bound Co2+ (which occupies the Mn2+ site on the enzyme) on the T1 of 31P from enzyme-bound 3',5'-pdTp and 5'-TMP, it was found that metal to 5'-phosphate distances are 0.9-1.6 A shorter in ternary complexes of the wild-type enzyme and of the D21E mutant than in ternary complexes of the D40G mutant. In all cases, the 5'-phosphate of pdTp is in the inner coordination sphere of Co2+ and the 3'-phosphate is predominantly in the second coordination sphere.     

Interaction of a spin-labeled analog of ATP with myosin

By means of spin labeled analogs of ATP we have shown that conformational changes in myosin molecule induced by variation of temperature take place in the region of the active centre. In case of Mg-ATP and unmodified myosin conformation of the active centre changes monotonously with the change in temperature but after the modification of S1 thiol groups by N-ethylmaleimide on the temperature dependence curve of rotational mobility of the spin label a discontinuous is observed at 14-16 degrees C. It is also observed in case of K+-EDTA-ATP, or Ca2+-ATP and unmodified myosin. It is shown that the chemical analogs of Mg2+-paramagnetic ion Mn2+ are directly connected with the myosin active centre in the presence of ATP(ADP), i. e. a triple complex enzyme-bivalent cation-substrate is formed.     

The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.     

Kinetic study on ESR signal decay of nitroxyl radicals,potent redox probes for in vivo ESR spectroscopy,caused by reactive oxygen species

The effect of the chemical structure of nitroxyl spin probes on the rate at which ESR signals are lost in the presence of reactive oxygen species (ROS) was examined. When the spin probes were reacted with either hydroxyl radical (.OH) or superoxide anion radical (O(2)(.-)) in the presence of cysteine or NADH, the probes lost ESR signal depending on both their ring structure and substituents. Pyrrolidine nitroxyl probes were relatively resistant to the signal decay caused by O(2)(.-) with cysteine/NADH. Signal decay rates for these reactions correlated with reported redox potentials of the nitroxyl/oxoammonium couple of spin probes, suggesting that the signal decay mechanism in both cases involves the oxidation of a nitroxyl group. The apparent rate constants of the reactions between the spin probe and .OH and between the spin probe and O(2)(.-) in the presence of cysteine were estimated using mannitol and superoxide dismutase (SOD), respectively, as competitive standards. The rate constants for spin probes and .OH were in the order of 10(9) M(-1) s(-1), much higher than those for the probes and O(2)(.-) in the presence of cysteine (10(3)-10(4) M(-1) s(-1)). These basic data are useful for the measurement of .OH and O(2)(.-) in living animals by in vivo ESR spectroscopy.     

Specific spin labelling of the sugar-H(+) symporter, GalP, in cell membranes of Escherichia coli: site mobility and overall rotational diffusion of the protein

The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, other membrane proteins were prereacted with N-ethylmaleimide in the presence of excess D-galactose to protect GalP. Inner membranes were then specifically spin labelled on Cys(374) of GalP with 4-maleimide-2,2,6,6-tetramethylpiperidine-1-oxyl. The electron paramagnetic resonance (EPR) spectra are characteristic of a single labelling site in which the mobility of the spin label is very highly constrained. This is confirmed with other nitroxyl spin labels, which are derivatives of iodoacetamide and indanedione. Saturation transfer EPR spectra indicate that the overall rotation of the GalP protein in the membrane is slow at low temperatures (approx. 2 degrees C), but considerably more rapid and highly anisotropic at physiological temperatures. The rate of rotation about the membrane normal at 37 degrees C is consistent with predictions for a 12-transmembrane helix assembly that is less than closely packed.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

Role of rapid movement of spin labels in interpreting EPR spectra for spin-labelled macromolecules

The method of spin labeling was used to monitor quick movements of side residues in protein monocrystals. The EPR spectra of monocrystals of spin-labeled lysozyme at different orientations of the tetrahonal crystal relative to the direction of the magnetic field were interpreted using the molecular dynamics method. A simple model was proposed, which enables one to calculate the trajectory of movements of the spin label by the molecular dynamic method over a relatively short period of time. The entire "frozen" protein molecule and a "defrozen" spin-labeled amino acid residue were considered in the framework of the model. To calculate the trajectories in vacuum, a model of spin-labeled lysozyme was constructed, and the parameters of force potentials for the atoms of the protein molecule and the spin label were specified. It follows from the calculations that the protein environment sterically hinders the range of eventual angular reorientations of the reporter NO-group of nitroxyl incorporated into the spin label, thereby affecting the shape of the EPR spectrum. However, the scatter in the positions of the reporter group in the angular space turned out to correspond to the Gauss distribution. Using the atomic coordinates of the spin label, obtained in a chosen time interval by the method of molecular dynamics, and taking into account the distribution of the states of the spin label in the ensemble of spin-labeled macromolecules in the crystal, we simulated the EPR spectra of monocrystals of spin-labeled lysozyme. The theoretical EPR spectra coincide well with the experimental.     

A spin label study of egg white avidin.

Avidin is a tetrametric protein (mass 68,000 daltons) that binds 4 molecules of vitamin biotin (1). The biotin binding sites, 1 per subunit, are grouped in two pairs at opposite ends of the avidin molecule (GREEN, N.M., KONIECZNY, L., TOMS, E.J., and VALENTINE, R.C. (1971) Biochem. J. 125, 781). We have studied the topography of the avidin binding sites with the aid of four spin-labeled analogs of biotin: 4-biotinamido-2,2,6,6-tetramethyl-1-piperidinyloxy (II), 3-biotinamido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (III), 3-biotinamidomethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IV), 4-(biotinylglycyl)-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (V). Fluorescence and optical absorption spectroscopy indicated that II to V occupied the same binding sites on avidin as did biotin. The electron spin resonance spectrum of the 4:1 complex between II and avidin contained broad line components characteristic of a highly immobilized spin label. Dipole-dipole interactions between spin labels bound to adjacent sites split each of the three major hyperfine lines into doublets with a separation of 13.8 G. The distance between adjacent bound nitroxide groups was calculated from this splitting to be 16 A. The dissociation of the 4:1 complex between II and avidin was biphasic with approximately half of the labels dissociating at a rate (kdiss equal to 2.51 times 10- minus 4 s- minus 1) that was much faster than the remainder (kdiss equal to 1.22 times 10- minus 5 s- minus 1). The electron spin resonance spectrum of the 2:1 complex between II and avidin clearly showed that, immediately after mixing, the spin labels were distributed in a random fashion among the available binding sites but that they slowly redistributed themselves so that each label bound to a site which was adjacent to an unoccupied site. The final time-independent electron spin resonance spectrum exhibited a splitting 69 G between the low and high field hyperfine lines which is characteristic of a highly immobilized, noninteracting spin label. Spin labels III and IV interacted with avidin in a similar fashion to that described for II with the exception that their dipolar splittings were 11.9 G and 14.2 G, respectively. From these splittings it was estimated that the distance between adjacent avidin-bound nitroxides was 16.7 A for labeled III and 15.7 A for label IV. The electron spin resonance spectrum of label V bound to avidin was characteristic of a noninteracting highly immobilized nitroxide with a maximum splitting of 62 G. The spectrum of V bound to avidin was independent of both time and the amount of bound label. The rate of dissociation of V from a 4:1 complex with avidin was monophasic. A model is proposed in which the recognition site for the heterocyclic ring system of biotin is represented as a cleft located within a hydrophobic depression in the surface of avidin.     

The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.     

Interspin distances in spin-labeled metmyoglobin variants determined by saturation recovery EPR

Saturation recovery (SR) electron paramagnetic resonance was used to determine the distance between iron and nitroxyl for spin-labeled metmyoglobin variants in low-spin and high-spin states of the Fe(III). The interspin distances were measured by analyzing the effect of the heme iron on the spin-lattice relaxation rates of the nitroxyl spin label using the modified Bloembergen equation for low-spin species, and an analogue of the Bloembergen equation for high-spin species. Insight simulations of the spin-labeled protein structures also were used to determine the interspin distances. The distances obtained by SR for high-spin and low-spin complexes with 15-20 A interspin distances, for low-spin CN(-) and high-spin formate adducts at distances up to about 30 A, and results from Insight calculations were in good agreement. For variants with 25-30 A interspin distances, the distances obtained by SR for the fluoride adducts were shorter than observed for the CN(-) or formate adducts or predicted by Insight simulations. Of the heme axial ligands examined (CN(-), imidazole, F(-), and formate), CN(-) is the best choice for determination of iron-nitroxyl distances in the range of 15-30 A.