Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin

The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy. Chemical shift perturbations and line broadening of amide resonances in the [(15)N,(1)H]HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms. The perturbed residues map over a large surface area for both proteins. For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex. In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain. These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin. A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.     

Study of the interaction of proflavine and isomeric diribonucleoside monophosphates CpG and GpC by proton magnetic resonance

A comparative study of the interaction of proflavine with isomeric diribonucleoside monophosphates CpG and GpC has been made by the method of 1H NMR (270 MHz). A method of calculation of the parameters of complex formation from the concentration dependences of proton chemical shifts of the dye has been proposed. The equilibrium constants of 1:1 and 1:2 complexes association of these molecules and the most probable structures of the complexes have been determined.     

Reactions of the four diastereomeric 16-amino-17-hydroxy-3-methoxy-estra-1,3,5(10)-trienes with aromatic ortho-hydroxy and heteroaromatic alpha-aldehydes and with 1, 3-dicarbonyl compounds-molecular structures of condensation products and of copper(II) complexes

Vicinal amino alcohols of steroids have been used as starting materials for the synthesis of chiral ligands with defined arrangements of functional groups. Condensation of the four diastereomeric 16,17-steroid amino alcohols 1a-1d with aromatic o-hydroxy and heteroaromatic alpha-aldehydes afforded the Schiff bases 2-6. When the 16,17-substituted compounds 2d, 5d, 6a, and 6d were in solution, the isomeric oxazolidines were detectable by (1)H NMR spectroscopy. The formation of oxazolidines could be avoided by using bulky aldehydes. Reduction of the Schiff bases (also in mixtures with oxazolidines) with NaBH(4) yielded the new N-substituted amino alcohols 12-15. The condensation products of 1a-1d with 1,3-dicarbonyl compounds (7 and 8) exhibited the 1-enamino-3-oxo structure ((1)H NMR spectroscopy). By means of X-ray analysis of 2a-2d, 3d, 7a, and 7c, the torsion angles for the 16N, 17O substituents, which are important for a participation of the 17O substituent in the complexation of metal ions, have been determined. Furthermore, a preferred arrangement between the chelate ring and the steroid plane existed in all investigated condensation products attributable to torsion angles 16H-C16-16N-C of 5-61 degrees. This arrangement was also preserved in the copper(II) complex 11 with 16alpha,17beta-trans configuration of the bidentate steroid ligand and a ratio of 2:1 for ligand: copper in contrast with dimeric copper(II) complexes with a tridentate steroid ligand of 16beta, 17beta-cis configuration (ratio of 1:1 for ligand:copper). The crystal structures of the condensation products are also discussed. In most cases, intermolecular hydrogen bonds between 17-hydroxy groups and the chelate oxygen caused polymeric strands.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

Complexes of Photosynthetic Redox Proteins Studied by NMR

In the photosynthetic redox chain, small electron transfer proteins shuttle electrons between the large membrane-associated redox complexes. Short-lived but specific protein:protein complexes are formed to enable fast electron transfer. Recent nuclear magnetic resonance (NMR) studies have elucidated the binding sites on plastocyanin, cytochrome c (6) and ferredoxin. Also the orientation of plastocyanin in complex with cytochrome f has been determined. Based on these results, general features that enable the formation of such transient complexes are discussed.     

The promotion of self-association of horse-heart cytochrome c by hexametaphosphate anions.

In the presence of the highly charged hexametaphosphate anion, horse heart cytochrome c aggregates to form stable protein complexes. The formation of protein aggregates has been detected by high-resolution 1H-NMR spectroscopy from an increase in the linewidth of resolved ferricytochrome c resonances with hexametaphosphate concentration. Alternatively, analytical ultracentrifugation reveals protein association from the increase in apparent sedimentation coefficients of cytochrome c in the presence of equimolar hexametaphosphate. Protein aggregation is dependent on the concentration of background electrolyte since in the range 10-150 mM sodium cacodylate alternative stabilisation of dimeric and trimeric complexes was observed by both NMR and analytical ultracentrifugation. A model is proposed for the mechanism of protein aggregation caused by polyphosphate binding to the surface of cytochrome c.     

Flavodoxin-cytochrome c interactions: circular dichroism and nuclear magnetic resonance studies

Circular dichroism and 1H and 31P nuclear magnetic resonance spectroscopy have been used to investigate complex formation between cytochrome c and the flavodoxins from Azotobacter vinelandii and Clostridium pasteurianum. Such complexes are known to be involved in the mechanism of electron transfer between these two redox proteins. A large increase in ellipticity in the Soret band of the cytochrome heme was observed upon formation of the Clostridium flavodoxin complex, whereas much smaller changes were found for the complexes with either Azotobacter flavodoxin or an 8 alpha-imidazolyl-FMN-substituted Clostridium flavodoxin analogue. Similarly, the magnitudes of the perturbations of the contact-shifted heme proton resonances obtained upon complexation of cytochrome c by Azotobacter flavodoxin were much smaller than those previously shown for Clostridium flavodoxin [Hazzard, J. T., & Tollin, G. (1985) Biochem. Biophys. Res. Commun. 130, 1281-1286]. 31P nuclear magnetic resonance measurements were also consistent with differences in the interactions between the components in the complexes of the two flavodoxins with cytochrome c. It is suggested that these spectral changes are due to a loosening or opening of the heme crevice upon Clostridium flavodoxin binding, which allows closer contact between the heme and flavin prosthetic groups and results in a faster rate of electron transfer. The implications of these observations for biological oxidation-reduction processes are considered.     

Model membrane morphology and crosslinking of oxidized lipids with proteins

Model membranes composed of thion-phosphatidylcholine, cardiolipin, and cytochrome c have been studied by 31P NMR, polyacrylamide gel electrophoresis, gel filtration, fluorescence, and freeze-fracturing. Covalent binding of oxidized phospholipids to cytochrome c was shown to result in the formation of high-molecular-weight oligomeric complexes via Schiff base formation between a protein molecule and aldehydes produced upon peroxidation of phospholipids. The initial steps of the protein oligomerization lead to the appearance of intramembranous particles (IMPs) of various size and distribution on freeze-fractured faces of these model membranes. In the final phase of the crosslinking between cytochrome c and oxidized products of cardiolipin there is a breakdown of membrane vesicles and formation of globular lipoprotein complexes which are seen as globular particles. It is believed that the covalent linking between the products of phospholipid peroxidation and membrane proteins causes the oligomerization of membrane proteins and structural alteration in the hydrophobic region of other models also and, perhaps, in biological membranes.     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

Preliminary 1H-NMR studies of the interaction between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway

The complex formation of two electron transfer proteins, cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway, has been shown by 1H-NMR spectroscopy. Presence of ferredoxin I produces ferricytochrome c3 1H-NMR spectrum modifications. The chemical shift of perturbated heme methyl resonances has been used to determine the stoichiometry of the complex. At pH 7.6 and 20 degrees C, the two proteins were found to form a complex 1:1 with an association constant, KA, of 10(4) M-1. Two of the four hemes are affected by presence of ferredoxin I and may be involved in the electron transfer sites. The heme methyl resonances are average resonances of free and bound cytochrome c3 resonances, indicating a fast exchange process on the NMR time scale.     

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions.

Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton NMR study of the cytochrome c:flavodoxin electron transfer complex

The effects of complex formation with flavodoxin on the proton NMR spectrum of cytochrome c are to change the resonance frequencies and to increase the bandwidths of most of the low and high field heme, Met-80, and His-18 protons. These effects are, in general, more pronounced than has been reported for other cytochrome c complexes. The degree of line broadening for many heme related resonances suggests that complex formation induces changes in the cytochrome structure. These results provide the first spectroscopic evidence which corroborates the proposed model for the cytochrome c: flavodoxin complex (1-3).     

Proton NMR characterization of isomeric sulfmyoglobins: preparation, interconversion, reactivity patterns, and structural features

The preparations of sulfmyoglobin (sulf-Mb) by standard procedures have been found heterogeneous by 1H NMR spectroscopy. Presented here are the results of a comprehensive study of the factors that influence the selection among the three dominant isomeric forms of sperm whale sulf-Mb and their resulting detailed optical and 1H NMR properties as related to their detectability and structural properties of the heme pocket. A single isomer is formed initially in the deoxy state; further treatment in any desired oxidation/ligation state can yield two other major isomers. Acid catalysis and chromatography facilitate formation of a second isomer, particularly in the high-spin state. At neutral pH, a third isomer is formed by a first-order process. The processes that alter oxidation/ligation state are found to be reversible and are judged to affect only the metal center, but the three isomeric sulf-Mbs are found to exhibit significantly different ligand affinity and chemical stability. The present results allow, for the first time, a rational approach for preparing a given isomeric sulf-Mb in an optimally pure state for subsequent characterization by other techniques. While optical spectroscopy can distinguish the alkaline forms, only 1H NMR clearly distinguishes all three ferric isomers. The ring current shifts in the carbonyl complexes of reduced sulf-Mb complexes support saturation for a pyrrole in each isomer. The hyperfine shift patterns in the various oxidation/spin states of sulf-Mbs indicate relatively small structural alteration, and the proximal and distal sides of the heme suggest that peripheral electronic effects are responsible for the differentially reduced ligand affinities for the three isomeric sulf-Mbs. The first 1H NMR spectra of sulfhemoglobins are presented, which indicate a structure similar to that of the initially formed sulf-Mb isomer but also suggest the presence of a similar molecular heterogeneity as found for sulf-Mb, albeit to a smaller extent.     

Electrostatic analysis of the interaction of cytochrome c with native and dimethyl ester heme substituted cytochrome b5

The stability of the complex formed between cytochrome c and dimethyl ester heme substituted cytochrome b5 (DME-cytochrome b5) has been determined under a variety of experimental conditions to evaluate the role of the cytochrome b5 heme propionate groups in the interaction of the two native proteins. Interaction between cytochrome c and the modified cytochrome b5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8 [25 degrees C (HEPPS), I = 5 mM], DME-cytochrome b5 and cytochrome c form a 1:1 complex with an association constant KA of 3 (1) X 10(6) M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DME-cytochrome b5 and cytochrome c is strongly dependent on ionic strength with KA ranging from 2.4 X 10(7) M-1 at I = 1 mM to 8.2 X 10(4) M-1 at I = 13 mM [pH 8.0 (HEPPS), 25 degrees C]. Calculations for the native, trypsin-solubilized form of cytochrome b5 and cytochrome c confirm that the intermolecular complex proposed by Salemme [Salemme, F. R. (1976) J. Mol. Biol. 102, 563] describes the protein-protein orientation that is electrostatically favored at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide

The 1:1 covalently cross-linked complex between horse cytochrome c and yeast cytochrome c peroxidase (ccp) has been formed by a slight modification of the method of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184-5190]. This earlier study has been extended to show that efficient cross-linking of the two proteins can occur in a variety of buffers over a broad ionic strength range. The substitution of ferrocytochrome c for ferricytochrome c in the cross-linking studies resulted in an increased yield of 1:1 complex (approximately 10-20%) under the conditions studied. An improved method for purifying the covalent complex in relatively large quantities is presented here as are the results of electrophoresis and proton NMR studies of the complex. Both electrophoresis and NMR studies indicate modification of some surface acidic amino acids in the covalent complex by the carbodiimide. The proton hyperfine-shifted resonances of cytochrome c are broadened in the covalent complex relative to free cytochrome c, and the resonances corresponding to the cytochrome c heme 3-CH3 and 8-CH3 groups are shifted closer together in the complex. Integration of NMR resonances confirms a 1:1 complex as the primary cross-linking reaction product. However, we also demonstrate that the covalent complex can be further coupled to ccp and to cytochrome c to form higher molecular weight aggregates.     

The type I/type II cytochrome c3 complex: an electron transfer link in the hydrogen-sulfate reduction pathway

In Desulfovibrio metabolism, periplasmic hydrogen oxidation is coupled to cytoplasmic sulfate reduction via transmembrane electron transfer complexes. Type II tetraheme cytochrome c3 (TpII-c3), nine-heme cytochrome c (9HcA) and 16-heme cytochrome c (HmcA) are periplasmic proteins associated to these membrane-bound redox complexes and exhibit analogous physiological function. Type I tetraheme cytochrome c3 (TpI-c3) is thought to act as a mediator for electron transfer from hydrogenase to these multihemic cytochromes. In the present work we have investigated Desulfovibrio africanus (Da) and Desulfovibrio vulgaris Hildenborough (DvH) TpI-c3/TpII-c3 complexes. Comparative kinetic experiments of Da TpI-c3 and TpII-c3 using electrochemistry confirm that TpI-c3 is much more efficient than TpII-c3 as an electron acceptor from hydrogenase (second order rate constant k = 9 x 10(8) M(-1) s(-1), K(m) = 0.5 microM as compared to k = 1.7 x 10(7) M(-1) s(-1), K(m) = 40 microM, for TpI-c3 and TpII-c3, respectively). The Da TpI-c3/TpII-c3 complex was characterized at low ionic strength by gel filtration, analytical ultracentrifugation and cross-linking experiments. The thermodynamic parameters were determined by isothermal calorimetry titrations. The formation of the complex is mainly driven by a positive entropy change (deltaS = 137(+/-7) J mol(-1) K(-1) and deltaH = 5.1(+/-1.3) kJ mol(-1)) and the value for the association constant is found to be (2.2(+/-0.5)) x 10(6) M(-1) at pH 5.5. Our thermodynamic results reveal that the net increase in enthalpy and entropy is dominantly produced by proton release in combination with water molecule exclusion. Electrostatic forces play an important role in stabilizing the complex between the two proteins, since no complex formation is detected at high ionic strength. The crystal structure of Da TpI-c3 has been solved at 1.5 angstroms resolution and structural models of the complex have been obtained by NMR and docking experiments. Similar experiments have been carried out on the DvH TpI-c3/TpII-c3 complex. In both complexes, heme IV of TpI-c3 faces heme I of TpII-c3 involving basic residues of TpI-c3 and acidic residues of TpII-c3. A secondary interacting site has been observed in the two complexes, involving heme II of Da TpII-c3 and heme III of DvH TpI-c3 giving rise to a TpI-c3/TpII-c3 molar ratio of 2:1 and 1:2 for Da and DvH complexes, respectively. The physiological significance of these alternative sites in multiheme cytochromes c is discussed.     

Complexes of aluminium(III) with glucose-6-phosphate in aqueous solutions

The interaction of aluminium(III) with glucose-6-phosphate (GP: LH2) in aqueous solutions has been studied from pH 1 to pH 8, by pH-potentiometry and multinuclear (31P, 27Al, 13C) NMR spectroscopy. Various mononuclear species (MLH2, MLH, ML, ML2H, ML2 and MLH(-3)) and dinuclear complexes M2L2H-n (n=1-4) are formed in the system. NMR clearly indicates that GP is already bound to Al(III) at pH 1. The potentiometric speciation results are confirmed and completed by spectroscopic experiments. Many peaks are observed in the 31P NMR spectra suggesting the formation of isomeric species. An attempt to assign the signals to the corresponding complexes is made, allowing a discussion about their structure. Interestingly enough no metal ion-induced deprotonation and coordination of the alcoholic-OH functions have been observed.     

Sedimentation equilibrium studies on the interaction between cytochrome c and cytochrome c peroxidase

The interaction between cytochrome c and cytochrome c peroxidase was investigated using sedimentation equilibrium at pH 6,20 degrees C, in a number of buffer systems varying in ionic strength between 1 and 100 mM. Between 10 and 100 mM ionic strengths, the sedimentation of the individual proteins was essentially ideal, and sedimentation equilibrium experiments on mixtures of the two proteins were analyzed assuming ideal solution behavior. Analysis of the distribution of mixtures of cytochrome c and cytochrome c peroxidase in the ultracentrifuge cell based on a model involving the formation of a 1:1 cytochrome c-cytochrome c peroxidase complex gave values of the equilibrium dissociation constant ranging from 2.3 +/- 2.7 microM at 10 mM ionic strength to infinity (no detectable interaction) at 100 mM ionic strength. Attempts to determine the presence of complexes involving two cytochrome c molecules bound to cytochrome c peroxidase were inconclusive.     

A modeling study of the interaction and electron transfer between cytochrome b 5 and some oxidized haemoglobins

Using Brownian motion simulations we have studied the formation of docked complexes of reduced cytochrome b5 and oxidized haemoglobin. Our results indicate that the presence of molecular electrostatic fields has a significant role to play in the formation of these complexes. In contrast to previous modeling studies on this system, we clearly identify electron transfer within an ensemble of similarly docked complexes rather than the formation of a single complex. Docking involves a number of acidic residues surrounding the exposed haem edge of cytochrome b5 and a set of basic residues surrounding the exposed haem edge of the globins. Although amino acids from the partner globin proteins are involved to a small extent in the binding of some of the complexes, the reactivity of any particular globin is essentially independent of the nature of its partner globin chain within the haemoglobin molecule. Comparison of results from adult and embryonic haemoglobins indicates a significant difference in complex formation. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes. These data provide a theoretical insight into the important process of re-reduction of oxidized haemoglobins as well as explaining the general inability to produce crystalline forms of many docked electron transfer complexes.