Structural rearrangement of human lymphotactin, a C chemokine, under physiological solution conditions

NMR spectra of human lymphotactin (hLtn), obtained under various solution conditions, have revealed that the protein undergoes a major conformational rearrangement dependent on temperature and salt concentration. At high salt (200 mm NaCl) and low temperature (10 degrees C), hLtn adopts a chemokine-like fold, which consists of a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix (Kulo?lu, E. S., McCaslin, D. R., Kitabwalla, M., Pauza, C. D., Markley, J. L., and Volkman, B. F. (2001) Biochemistry 40, 12486-12496). We have used NMR spectroscopy, sedimentation equilibrium, and intrinsic fluorescence to monitor the reversible conformational change undergone by hLtn as a function of temperature and ionic strength. We have used two-, three- and four-dimensional NMR spectroscopy of isotopically enriched protein samples to determine structural properties of the conformational state stabilized at 45 degrees C and 0 mm NaCl. Patterns of NOEs and (1)H(alpha) and (13)C chemical shifts show that hLtn rearranges under these conditions to form a four-stranded, antiparallel beta-sheet with a pattern of hydrogen bonding that is completely different from that of the chemokine fold stabilized at 10 degrees C and 200 mm NaCl. The C-terminal alpha-helix observed at 10 degrees C and 200 mm NaCl, which is conserved in other chemokines, is absent at 45 degrees C and no salt, and the last 38 residues of the protein are completely disordered, as indicated by heteronuclear (15)N-(1)H NOEs. Temperature dependence of the tryptophan fluorescence of hLtn in low and high salt confirmed that the chemokine conformation is stabilized by increased ionic strength. Sedimentation equilibrium analytical ultracentrifugation showed that hLtn at 40 degrees C in the presence of 100 mm NaCl exists mainly as a dimer. Under near physiological conditions of temperature, pH, and ionic strength, both the chemokine-like and non-chemokine-like conformations of hLtn are significantly populated. The functional relevance of this structural interconversion remains to be elucidated.     

Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein.

The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the absence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(-1) M(-1). Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS. Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein [Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry 38, 1018-1029]. Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif.     

Structural characterization of protein kinase A as a function of nucleotide binding. Hydrogen-deuterium exchange studies using matrix-assisted laser desorption ionization-time of flight mass spectrometry detection

Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J., and Adams, J. A. (1999) Biochemistry 38, 12072-12079). To assess whether specific structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.     

pH-Dependent Conformational Changes in Bacterial Hsp90 Reveal a Grp94-Like Conformation at pH 6 That Is Highly Active in Suppression of Citrate Synthase Aggregation

The molecular chaperone Hsp90 depends upon large conformational rearrangements for its function. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, structural and kinetic studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shift the equilibrium between a preexisting set of conformational states in an organism-dependent manner. While many conformations of Hsp90 have been described, little is known about how they relate to chaperone function. In this study, we show that the conformational equilibrium of the bacterial Hsp90, HtpG, can be shifted with pH. Using small-angle X-ray scattering, we identify a two-state pH-dependent conformational equilibrium for apo HtpG. Our structural modeling reveals that this equilibrium is observed between the previously observed extended state and a second state that is strikingly similar to the recently solved Grp94 crystal structure. In the presence of nonhydrolyzable 5′-adenylyl-β,γ-imidodiphosphate, a third state, which is identical with the solved AMPPNP-bound structure from yeast Hsp90, is populated. Electron microscopy confirmed the observed conformational equilibria. We also identify key histidine residues that control this pH-dependent equilibrium; using mutagenesis, we successfully modulate the conformational equilibrium at neutral pH. Using these mutations, we show that the Grp94-like state provides stronger aggregation protection compared to the extended apo conformation in the context of a citrate synthase aggregation assay. These studies provide a more detailed view of HtpG's conformational dynamics and provide the first linkage between a specific conformation and chaperone function.     

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].     

Temperature dependent mechanical unfolding and refolding of a protein studied by thermo-regulated optical tweezers

Temperature is a useful system variable to gather kinetic and thermodynamic information from proteins. Usually, free energy and the associated entropic and enthalpic contributions are obtained by quantifying the conformational equilibrium based on melting experiments performed in bulk conditions. Such experiments are suitable only for those small single-domain proteins whose side reactions of irreversible aggregation are unlikely to occur. Here, we avoid aggregation by pulling single-protein molecules in a thermo-regulated optical tweezers. Thus, we are able to explore the temperature dependence of the thermodynamic and kinetic parameters of MJ0366 from Methanocaldococcus jannaschii at the single-molecule level. By performing force-ramp experiments between 2°C and 40°C, we found that MJ0366 has a nonlinear dependence of free energy with temperature and a specific heat change of 2.3 ± 1.2 kcal/mol¹K. These thermodynamic parameters are compatible with a two-state unfolding/refolding mechanism for MJ0366. However, the kinetics measured as a function of the temperature show a complex behavior, suggesting a three-state folding mechanism comprising a high-energy intermediate state. The combination of two perturbations, temperature and force, reveals a high-energy species in the folding mechanism of MJ0366 not detected in force-ramp experiments at constant temperature.     

Slow folding of a three-helix protein via a compact intermediate

The KIX domain of CREB binding protein (CBP) forms a small three-helix bundle which folds autonomously. Previous equilibrium unfolding experiments led to the suggestion that folding may not be strictly two-state. To investigate the folding mechanism in more detail, the folding kinetics of KIX have been studied by urea jump fluorescence-detected stopped-flow experiments. Clear evidence for an intermediate is obtained from the plot of the natural log of the observed rate constant versus denaturant concentration, the chevron plot, and from analysis of the initial fluorescence amplitudes of the stopped-flow experiments. The chevron plot exhibits a change in shape, rollover, at low denaturant concentrations, characteristic of the formation of an intermediate. The kinetic data can be fit to a three-state model involving a compact intermediate. An on-pathway model predicts that the position of the intermediate lies close to the native state. The folding rate in the absence of denaturant is 260 s(-)(1) at pH 7.5 and 25 degrees C. This is significantly slower than the rates of other helical proteins similar in size. The slow folding may be due to the necessity of forming a buried polar interaction in the native state. The potential functional significance of the folding intermediate is discussed.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Folding and stability of the extracellular domain of the human amyloid precursor protein

The beta-amyloid peptide (A beta), the major component of the senile plaques found in the brains of Alzheimer's disease patients, is derived from proteolytic processing of a transmembrane glycoprotein known as the amyloid precursor protein (APP). Human APP exists in various isoforms, of which the major ones contain 695, 751, and 770 amino acids. Proteolytic cleavage of APP by alpha- or beta-secretases releases the extracellular soluble fragments sAPP alpha or sAPP beta, respectively. Despite the fact that sAPP alpha plays important roles in both physiological and pathological processes in the brain, very little is known about its structure and stability. We have recently presented a structural model of sAPP alpha 695 obtained from small-angle x-ray scattering measurements (Gralle, M., Botelho, M. M., Oliveira, C. L. P., Torriani, I., and Ferreira, S. T. (2002) Biophys. J. 83, 3513-3524). We now report studies on the folding and stabilities of sAPP alpha 695 and sAPP alpha 770. The combined use of intrinsic fluorescence, 4-4'-Dianilino-1,1'binaphthyl-5,5'-disulfonic acid (bis-ANS) fluorescence, circular dichroism, differential ultraviolet absorption, and small-angle x-ray scattering measurements of the equilibrium unfolding of sAPP alpha 695 and sAPP alpha 770 by GdnHCl and urea revealed multistep folding pathways for both sAPP alpha isoforms. Such stepwise folding processes may be related to the identification of distinct structural domains in the three-dimensional model of sAPP alpha. Furthermore, the relatively low stability of the native state of sAPP alpha suggests that conformational plasticity may play a role in allowing APP to interact with a number of distinct physiological ligands.     

The conformation of the T-antigen disaccharide bound to Maclura pomifera agglutinin in aqueous solution

The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (beta-d-Gal-(1-->3)-alpha-d-GalNAc-(1-->O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z. , and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the beta-(1-->3)glycosidic linkage in the solution complex with interglycosidic angles phi/psi = 45/-65 degrees and -65/-18 degrees. These differ from the bound conformation of the ligand in the crystal (phi/psi = 39/-8 degrees ) and are not highly populated by the ligand in the free state. The reason for the structural differences at the beta-(1-->3)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.     

Effects of primary sequence differences on the global structure and function of an enzyme: a study of pyruvate kinase isozymes

Pyruvate kinase is an important glycolytic enzyme which is expressed differentially as four distinct isozymes whose catalytic activity is regulated in a tissue-specific manner. The kidney isozyme is known to exhibit sigmoidal kinetics, whereas the muscle isozyme exhibits hyperbolic kinetic properties. By integration of the crystallographic [Stuart, D. I., Levine, M., Muirhead, H., & Stammers, D.K. (1979) J. Mol. Biol. 134, 109-142] and primary sequence data [Noguchi, T., Inoue, H., & Tanaka, T. (1986) J. Biol. Chem. 261, 13807], it was shown that the primary sequence for the C alpha 1 and C alpha 2 regions may constitute the allosteric switching site. To provide insights into the effects of the localized sequence change on the global structural and functional behavior of the enzyme, kinetic studies under a wide spectrum of conditions were conducted for both the muscle and kidney isozymes. These conditions include measurements of enzyme activity as a function of substrate concentrations with different concentrations of allosteric inhibitors or activators. These results showed that both isozymes exhibit the same regulatory properties although quantitatively the distribution of active and inactive forms and the various dissociation constants which govern the binding of substrate and allosteric effectors with the enzyme are different. For such a majority of equilibrium constants to be altered, the localized primary sequence change must confer global perturbations which are manifested as differences in the various equilibrium constants. Structural information about these two isozymes was provided by phase-modulation measurement of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Side-chain interactions in the folding pathway of a Fyn SH3 domain mutant studied by relaxation dispersion NMR spectroscopy

A major challenge to the study of protein folding is the fact that intermediate states along the reaction pathway are generally unstable and thus difficult to observe. Recently developed NMR relaxation dispersion experiments present an avenue to accessing such states, providing kinetic, thermodynamic, and structural information for intermediates with small (greater than or equal to approximately 1%) populations at equilibrium. We have employed these techniques to study the three-state folding reaction of the G48M Fyn SH3 domain. Using (13)C-, (1)H-, and (15)N-based methods, we have characterized backbone and side-chain interactions in the folded, unfolded, intermediate, and transition states, thereby mapping the energy landscape of the protein. We find that the intermediate, populated to approximately 1%, contains nativelike structure in a central beta-sheet, and is disordered at the amino and carboxy termini. The intermediate is stabilized by side-chain van der Waals contacts, yet (13)C chemical shifts indicate that methyl-containing residues remain disordered. This state has a partially structured backbone and a collapsed yet mobile hydrophobic core and thus closely resembles a molten globule. Nonpolar side-chain contacts are formed in the unfolded-intermediate transition state; these interactions are disrupted in the intermediate-folded transition state, possibly allowing side chains to rearrange as they adopt the native packing configuration. This work illustrates the power of novel relaxation dispersion experiments in characterizing excited states that are "invisible" in even the most sensitive of NMR experiments.     

Cooperative unfolding of a metastable serpin to a molten globule suggests a link between functional and folding energy landscapes

Alpha-1 antitrypsin (alpha(1)-AT) is a member of the serpin class of protease inhibitors, and folds to a metastable state rather than its thermodynamically most stable native state. Upon cleavage by a target protease, alpha(1)-AT undergoes a dramatic conformational change to a stable form, translocating the bound protease more than 70 A to form an inhibitory protease-serpin complex. Numerous mutagenesis studies on serpins have demonstrated the trade-off between the stability of the metastable state on the one hand and the inhibitory efficiency on the other. Studies of the equilibrium unfolding of serpins provide insight into this connection between structural plasticity and metastability. We studied equilibrium unfolding of wild-type alpha(1)-AT using hydrogen-deuterium/exchange mass spectrometry to characterize the structure and the stability of an equilibrium intermediate that was observed in low concentrations of denaturant in earlier studies. Our results show that the intermediate observed at low concentrations of denaturant has no protection from hydrogen-deuterium exchange, indicating a lack of stable structure. Further, differential scanning calorimetry of alpha(1)-AT at low concentrations of denaturant shows no heat capacity peak during thermal denaturation, indicating that the transition from the intermediate to the unfolded state is not a cooperative first-order-like phase transition.. Our results show that the unfolding of alpha(1)-AT involves a cooperative transition to a molten globule form, followed by a non-cooperative transition to a random-coil form as more guanidine is added. Thus, the entire alpha(1)-AT molecule consists of one cooperative structural unit rather than multiple structural domains with different stabilities. Furthermore, our results together with previous mutagenesis studies suggest a possible link between an equilibrium molten globule and a functional intermediate that may be populated during the protease inhibition.     

Oligomeric States along the Folding Pathways of β2-Microglobulin: Kinetics,Thermodynamics, and Structure

The transition of proteins from their soluble functional state to amyloid fibrils and aggregates is associated with the onset of several human diseases. Protein aggregation often requires some structural reshaping and the subsequent formation of intermolecular contacts. Therefore, the study of the conformation of excited protein states and their ability to form oligomers is of primary importance for understanding the molecular basis of amyloid fibril formation. Here, we investigated the oligomerization processes that occur along the folding of the amyloidogenic human protein β2-microglobulin. The combination of real-time two-dimensional NMR data with real-time small-angle X-ray scattering measurements allowed us to derive thermodynamic and kinetic information on protein oligomerization of different conformational states populated along the folding pathways. In particular, we could demonstrate that a long-lived folding intermediate (I-state) has a higher propensity to oligomerize compared to the native state. Our data agree well with a simple five-state kinetic model that involves only monomeric and dimeric species. The dimers have an elongated shape with the dimerization interface located at the apical side of β2-microglobulin close to Pro32, the residue that has a trans conformation in the I-state and a cis conformation in the native (N) state. Our experimental data suggest that partial unfolding in the apical half of the protein close to Pro32 leads to an excited state conformation with enhanced propensity for oligomerization. This excited state becomes more populated in the transient I-state due to the destabilization of the native conformation by the trans-Pro32 configuration.     

Conformational states of human rat sarcoma (Ras) protein complexed with its natural ligand GTP and their role for effector interaction and GTP hydrolysis

The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K(12) is 11.3. K(12) of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)·Mg(2+)·GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.     

The structural and functional role of lysine residues in the binding domain of cytochrome c in the electron transfer to cytochrome c oxidase.

The interactions of yeast iso-1 cytochrome c with bovine cytochrome c oxidase were studied using cytochrome c variants in which lysines of the binding domain were substituted by alanines. Resonance Raman spectra of the fully oxidized complexes of both proteins reveal structural changes of both the heme c and the hemes a and a3. The structural changes in cytochrome c are the same as those observed upon binding to phospholipid vesicles where the bound protein exists in two conformers, B1 and B2. Whereas the structure of B1 is the same as that of the unbound cytochrome c, the formation of B2 is associated with substantial alterations of the heme pocket. In cytochrome c oxidase, the structural changes in both hemes refer to more subtle perturbations of the immediate protein environment and may be a result of a conformational equilibrium involving two states. These changes are qualitatively different to those observed for cytochrome c oxidase upon poly-l-lysine binding. The resonance Raman spectra of the various cytochrome c/cytochrome c oxidase complexes were analyzed quantitatively. The spectroscopic studies were paralleled by steady-state kinetic measurements of the same protein combinations. The results of the spectra analysis and the kinetic studies were used to determine the stability of the complexes and the conformational equilibria B2/B1 for all cytochrome c variants. The complex stability decreases in the order: wild-type WT > J72K > K79A > K73A > K87A > J72A > K86A > K73A/K79A (where J is the natural trimethyl lysine). This order is not exhibited by the conformational equilibria. The electrostatic control of state B2 formation does not depend on individual intermolecular salt bridges, but on the charge distribution in a specific region of the front surface of cytochrome c that is defined by the lysyl residues at positions 72, 73 and 79. On the other hand, the conformational changes in cytochrome c oxidase were found to be independent of the identity of the bound cytochrome c variant. The maximum rate constants determined from steady-state kinetic measurements could be related to the conformational equilibria of the bound cytochrome c using a simple model that assumes that the conformational transitions are faster than product formation. Within this model, the data analysis leads to the conclusion that the interprotein electron transfer rate constant is around two times higher in state B2 than in B1. These results can be interpreted in terms of an increase of the driving force in state B2 as a result of the large negative shift of the reduction potential.