RESUMEN
Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.
Asunto(s)
Azurina , Metaloproteínas , Azurina/química , Cobre/química , Metaloproteínas/metabolismo , Disulfuros/química , Temperatura , Iones , Pliegue de ProteínaRESUMEN
"What I cannot create, I do not understand"âRichard Feynman. This sentiment motivates the entire field of artificial metalloenzymes. Naturally occurring enzymes catalyze reactions with efficiencies, rates, and selectivity that generally cannot be achieved in synthetic systems. Many of these processes represent vital building blocks for a sustainable society, including CO2 conversion, nitrogen fixation, water oxidation, and liquid fuel synthesis. Our inability as chemists to fully reproduce the functionality of naturally occurring enzymes implicates yet-unknown contributors to reactivity. To identify these properties, it is necessary to consider all of the components of naturally occurring metalloenzymes, from the active site metal(s) to large-scale dynamics. In this Account, we describe the holistic development of a metalloprotein-based model that functionally reproduces the acetyl coenzyme A synthase (ACS) enzyme.ACS catalyzes the synthesis of a thioester, acetyl coenzyme A, from gaseous carbon monoxide, a methyl group donated by a cobalt corrinoid protein, and coenzyme A. The active site of ACS contains a bimetallic nickel site coupled to a [4Fe-4S] cluster. This reaction mimics Monsanto's acetic acid synthesis and represents an ancient process for incorporating inorganic carbon into cellular biomass through the primordial Wood-Ljungdahl metabolic pathway. From a sustainability standpoint, the reversible conversion of C1 substrates into an acetyl group and selective downstream transfer to a thiolate nucleophile offer opportunities to expand this reactivity to the anthropogenic synthesis of liquid fuels. However, substantial gaps in our understanding of the ACS catalytic mechanism coupled with the enzyme's oxygen sensitivity and general instability have limited these applications. It is our hope that development of an artificial metalloenzyme that carries out ACS-like reactions will advance our mechanistic understanding and enable synthesis of robust compounds with the capacity for similar reactivity.To construct this model, we first focused on the catalytic proximal nickel (NiP) site, which has a single metal center bound by three bridging cysteine residues in a "Y"-shaped arrangement. With an initial emphasis on reproducing the general structure of a low-coordinate metal binding site, the type I cupredoxin, azurin, was selected as the protein scaffold, and a nickel center was incorporated into the mononuclear site. Using numerous spectroscopic and computational techniques, including electron paramagnetic resonance (EPR) spectroscopy, nickel-substituted azurin was shown to have similar electronic and geometric structures to the NiP center in ACS. A substrate access channel was installed, and both carbon monoxide and a methyl group were shown to bind individually to the reduced NiI center. The elusive EPR-active S = 1/2 Ni-CH3 species, which has never been detected in native ACS, was observed in the azurin-based model, establishing the capacity of a biological NiI species to support two-electron organometallic reactions. Pulsed EPR studies on the S = 1/2 Ni-CH3 species in azurin suggested a noncanonical electronic structure with an inverted ligand field, which was proposed to prevent irreversible site degradation. This model azurin protein was ultimately shown to perform carbon-carbon and carbon-sulfur bond formation using sequential, ordered substrate addition for selective, stoichiometric thioester synthesis. X-ray spectroscopic methods were used to provide characterization of the remaining catalytic intermediates, resolving some debate over key mechanistic details.The overall approach and strategies that we employed for the successful construction of a functional protein-based model of ACS are described in this Account. We anticipate that these principles can be adapted across diverse metalloenzyme classes, providing essential mechanistic details and guiding the development of next-generation, functional artificial metalloenzymes.
Asunto(s)
Azurina , Metaloproteínas , Azurina/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Níquel/química , Monóxido de Carbono/metabolismo , Espectroscopía de Resonancia por Spin del ElectrónRESUMEN
Azurin which is a bacterial secondary metabolite has attracted much attention as potential anticancer agent in recent years. This copper-containing periplasmic redox protein supresses the tumor growth selectively. High-level secretion of proteins into the culture medium offers a significant advantage over periplasmic or cytoplasmic expression. The aim of this study was to investigate the effect of nonionic surfactants on the expression of the Pseudomonas aeruginosa azurin. Different concentrations of Triton X-100 and Tween 80 were used as supplements in growth media and extracellular azurin production was stimulated by both surfactants. According to western blot analysis results, in the presence of Triton X-100, maximum azurin expression level was achieved with 96 h of incubation at 1% concentration, and 48 h at 2% concentration. On the other hand, maximum azurin expression level was achieved in the presence of 1% Tween 80 at 72 h incubation. This study suggested for the first time a high level of azurin secretion from P. aeruginosa in the presence of Triton X-100 or Tween 80, which would be advantageous for the purification procedure.
Asunto(s)
Azurina , Azurina/análisis , Azurina/metabolismo , Proteínas Bacterianas/metabolismo , Cobre/metabolismo , Octoxinol/farmacología , Polisorbatos/metabolismo , Polisorbatos/farmacología , Pseudomonas aeruginosa/metabolismoRESUMEN
The use of live bacteria in cancer therapies offers exciting possibilities. Nowadays, an increasing number of genetically engineered bacteria are emerging in the field, with applications both in therapy and diagnosis. In parallel, purified bacterial products are also gaining relevance as new classes of bioactive products to treat and prevent cancer growth and metastasis. In the first part of the article, we review the latest findings regarding the use of live bacteria and products as anti-cancer agents, paying special attention to immunotoxins, proteins, and peptides. In particular, we focus on the recent results of using azurin or its derived peptide as anticancer therapeutic agents. In the second part, we discuss the challenges of using metagenomic techniques as a distinctive approach for discovering new anti-cancer agents from bacterial origin.
Asunto(s)
Antineoplásicos/metabolismo , Bacterias/metabolismo , Terapia Biológica/métodos , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Neoplasias/diagnóstico , Neoplasias/terapia , Antineoplásicos/aislamiento & purificación , Antineoplásicos/uso terapéutico , Azurina/metabolismo , Bacterias/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Biotecnología/métodos , Biología Computacional , Inmunotoxinas/metabolismo , Metagenómica , Tecnología Farmacéutica/métodosRESUMEN
Hard-ligand, high-potential copper sites have been characterized in double mutants of Pseudomonas aeruginosa azurin (C112D/M121X (X = L, F, I)). These sites feature a small A(zz)(Cu) splitting in the EPR spectrum together with enhanced electron transfer activity. Due to these unique properties, these constructs have been called "type zero" copper sites. In contrast, the single mutant, C112D, features a large A(zz)(Cu) value characteristic of the typical type 2 Cu(II). In general, A(zz)(Cu) comprises contributions from Fermi contact, spin dipolar, and orbital dipolar terms. In order to understand the origin of the low A(zz)(Cu) value of type zero Cu(II), we explored in detail its degree of covalency, as manifested by spin delocalization over its ligands, which affects A(zz)(Cu) through the Fermi contact and spin dipolar contributions. This was achieved by the application of several complementary EPR hyperfine spectroscopic techniques at X- and W-band (â¼9.5 and 95 GHz, respectively) frequencies to map the ligand hyperfine couplings. Our results show that spin delocalization over the ligands in type zero Cu(II) is different from that of type 2 Cu(II) in the single C112D mutant. The (14)N hyperfine couplings of the coordinated histidine nitrogens are smaller by about 25-40%, whereas that of the (13)C carboxylate of D112 is about 50% larger. From this comparison, we concluded that the spin delocalization of type zero copper over its ligands is not dramatically larger than in type 2 C112D. Therefore, the reduced A(zz)(Cu) value of type zero Cu(II) is largely attributable to an increased orbital dipolar contribution that is related to its larger g(zz) value, as a consequence of the distorted tetrahedral geometry. The increased spin delocalization over the D112 carboxylate in type zero mutants compared to type 2 C112D suggests that electron transfer paths involving this residue are enhanced.
Asunto(s)
Azurina/química , Cobre/química , Electrones , Pseudomonas aeruginosa/química , Ácido Aspártico/química , Ácido Aspártico/genética , Azurina/genética , Azurina/metabolismo , Cisteína/química , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Escherichia coli , Histidina/química , Leucina/química , Leucina/genética , Ligandos , Espectroscopía de Resonancia Magnética , Metionina/química , Metionina/genética , Modelos Moleculares , Mutación , Oxidación-Reducción , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
PURPOSE: Characterize the preclinical pharmacokinetics, metabolic profile, multi-species toxicology, and antitumor efficacy of azurin-p28 (NSC 745104), an amphipathic, 28 amino acid fragment (aa 50-77) of the copper containing redox protein azurin that preferentially enters cancer cells and is currently under development for treatment of p53-positive solid tumors. METHODS: An LC/MS/MS assay was developed, validated, and applied to liver microsomes, serum, and tumor cells to assess cellular uptake and metabolic stability. Pharmacokinetics was established after administration of a single intravenous dose of p28 in preclinical species undergoing chronic toxicity testing. Antitumor efficacy was assessed on human tumor xenografts. A human therapeutic dose was predicted based on efficacy and pharmacokinetic parameters. RESULTS: p28 is stable, showed tumor penetration consistent with selective entry into tumor cells and significantly inhibited p53-positive tumor growth. Renal clearance, volume of distribution, and metabolic profile of p28 was relatively similar among species. p28 was non-immunogenic and non-toxic in mice and non-human primates (NHP). The no observed adverse effect level (NOAEL) was 120 mg/kg iv in female mice. A NOAEL was not established for male mice due to decreased heart and thymus weights that was reversible and did not result in limiting toxicity. In contrast, the NOAEL for p28 in NHP was defined as the highest dose (120 mg/kg/dose; 1,440 mg/m(2)/dose) studied. The maximum-tolerated dose (MTD) for subchronic administration of p28 to mice is >240 mg/kg/dose (720 mg/m(2)/dose), while the MTD for subchronic administration of p28 to Cynomolgous sp. is >120 mg/kg (1,440 mg/m(2)/dose). The efficacious (murine) dose of p28 was 10 mg/kg ip per day. CONCLUSIONS: p28 does not exhibit preclinical immunogenicity or toxicity, has a similar metabolic profile among species, and is therapeutic in xenograft models.
Asunto(s)
Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Azurina/efectos adversos , Azurina/farmacocinética , Neoplasias/tratamiento farmacológico , Fragmentos de Péptidos/farmacocinética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Azurina/metabolismo , Azurina/uso terapéutico , Biotransformación , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Ratones Desnudos , Nivel sin Efectos Adversos Observados , Fragmentos de Péptidos/efectos adversos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/uso terapéutico , Organismos Libres de Patógenos Específicos , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for "acidophile CcO partner," which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu(A) center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.
Asunto(s)
Acidithiobacillus/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Azurina/química , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis , Concentración de Iones de Hidrógeno , Espectrometría de Masas/métodos , Metaloproteínas/química , Metaloproteínas/genética , Modelos Biológicos , Oxidación-Reducción , Consumo de Oxígeno , Unión Proteica , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.
Asunto(s)
Azurina/química , Apoproteínas/química , Azurina/genética , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cristalografía por Rayos X , Cisteína/metabolismo , Mutación , Oxidación-Reducción , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Pseudomonas aeruginosa , TemperaturaRESUMEN
Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe(2+) medium (pH 2.5) supplemented with 6 microM Hg(2+). In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 microM Hg(2+). When incubated for 3 h in a salt solution (pH 2.5) with 0.7 microM Hg(2+), resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe(2+) was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30 degrees C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe(2+)-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 microM Hg(2+) and 1 mM Fe(2+), plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe(2+)-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe(2+)-dependent mercury volatilization activity of the plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , Compuestos Ferrosos/metabolismo , Mercurio/metabolismo , Thiobacillus/metabolismo , Azurina/análogos & derivados , Azurina/farmacología , Medios de Cultivo , Farmacorresistencia Microbiana , Mercurio/farmacología , Cianuro de Sodio/farmacología , Thiobacillus/efectos de los fármacos , Thiobacillus/crecimiento & desarrollo , VolatilizaciónRESUMEN
A negatively photo-regulated gene (bcb) has been isolated by means of differential hybridization of a genomic library of Arabidopsis thaliana. In mature plants, a 20-fold increase in the amount of steady-state bcb mRNA can be detected upon 48 h of dark adaption. The expression level of the gene is also dependent upon the developmental stage of the plant. The 21.5-kDa gene product (BCB) shows extensive similarity with blue Cu(2+)-binding proteins such as plastocyanin and stellacyanin. The protein abundance increases only twofold upon dark adaption, which implies the presence of post-transcriptional control. The isolation of a novel negatively photoregulated gene allows us to investigate the complex expression profile of genes responding to the absence of light.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Azurina/análogos & derivados , Proteínas Portadoras/genética , Cobre , Regulación de la Expresión Génica , Luz , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Azurina/genética , Secuencia de Bases , ADN Complementario , Regulación de la Expresión Génica/efectos de la radiación , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Time-resolved fluorescence spectroscopy permits the direct assessment of proteins motions in the picosecond-nanosecond time-scaled, i.e., in a time-window compatible with observation of relevant motions of the protein matrix. The intrinsic fluorescence emission from tryptophan and tyrosine residues provides a convenient tool to follow these dynamic events in proteins. In the present investigation, the use of time-resolved fluorescence spectroscopy to monitor protein dynamics is illustrated by a study of the effects of temperature and calcium binding on the internal dynamics of the calcium-binding protein, parvalbumin, and by an investigation of the effects of hydration on the measurements of both fluorescence intensity and anisotropy decays provided complementary information regarding the flexibility of aromatic side chains in the proteins investigated, which could be correlated with environmental effects on protein structure.
Asunto(s)
Parvalbúminas/química , Azurina/química , Calcio/química , Calcio/metabolismo , Muramidasa/química , Parvalbúminas/metabolismo , Espectrometría de Fluorescencia , Temperatura , Triptófano/químicaRESUMEN
Methylomonas J is an obligate methylotroph although it is unable to grow on methane. Like Pseudomonas AM1, it produces two blue copper proteins when growing on methylamine, one of which is the recipient of electrons from the methylamine dehydrogenase. When grown on methanol, only the other blue copper protein is produced. We have determined the amino acid sequences of these blue copper proteins, and show that they are both true azurins. The sequences are clearly homologous to those of the proteins characterized from fluorescent pseudomonads and various species of Alcaligenes, and can be aligned with them and with each other without the need to postulate any internal insertions or deletions in the sequences. The iso-1 azurin, the one produced during both methanol and methylamine growth, shows 59-65% identity with these other azurins, whereas the iso-2 protein shows only 47-53% identity. The proteins show 52% identity with each other. The two functionally equivalent blue copper proteins from Pseudomonas AM1 belong to two sequence classes that are quite distinct from the true azurins. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50151 (23 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.
Asunto(s)
Azurina , Proteínas Bacterianas , Methylococcaceae/metabolismo , Secuencia de Aminoácidos , Transporte de Electrón , Datos de Secuencia MolecularRESUMEN
The amino acid sequences of two blue copper proteins from the pink facultative methylotroph Pseudomonas AM1 (N.C.I.B. 9133) were determined. They each consist of a single polypeptide chain and bind one copper atom. Amicyanin contains 99 and pseudoazurin 123 residues. Copper-binding sites, consisting of the side chains of two histidine, one cysteine and one methionine residues, can be recognized in each protein by analogy with azurin and plastocyanin, but the spacings of the ligand residues are different, and other sequence similarity is limited. Proteins that are in the pseudoazurin sequence class can be recognized in some strains of Alcaligenes, and probably also in Paracoccus denitrificans. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50130 (23 pp.) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1985) 225, 5.
Asunto(s)
Azurina , Proteínas Bacterianas , Metaloproteínas , Pseudomonas/análisis , Secuencia de Aminoácidos , Azurina/análogos & derivadosRESUMEN
The production and spectroscopic properties of an L-selenomethionine-containing homolog of Pseudomonas aeruginosa azurin are described. The amino acid substitution was carried out by developing an L-methionine-dependent bacterial strain from a fully functional ATCC culture. Uptake studies monitored using L-[75Se]methionine indicated that L-selenomethionine was incorporated into the protein synthetic pathway of Pseudomonas bacteria in a manner analogous to L-methionine. Several batches of bacteria were grown, and one sample of isolated and purified selenoazurin (azurin in which methionine was substituted by selenomethionine) was found (by neutron activation analysis) to contain 5.2 +/- 0.8 seleniums/copper. Correspondingly, a residual 0.35 methionines, relative to 6.0 in the native protein, were found by amino acid analysis in this azurin sample. The redox potential and extinction coefficient of this selenoazurin were found to be 333 +/- 1 mV (pH 7.0, I = 0.22) and 5855 +/- 160 M-1 cm-1 at 626 +/- 1 nm, respectively. Visible electronic, CD, and EPR spectra are reported and Gaussian curve fitting to the former spectrum allowed assignment of the selenomethionine Se----Cu(II) transition to a band found at 18034 cm-1, based upon an observed 450 cm-1 shift to the red from the analogous band position in the native protein. The data are consistent with a relatively more covalent copper site stabilizing the reduced, Cu(I), form in the selenoprotein. A role for the methionine as a modulator of the blue copper site redox potential by metal----ligand back bonding from Cu(I) is discussed in terms of a ligand sphere which limits the valence change at copper to much less than 1 during a redox cycle.
Asunto(s)
Azurina/análisis , Proteínas Bacterianas/análisis , Pseudomonas aeruginosa/análisis , Selenio/análisis , Selenometionina/análisis , Dicroismo Circular , Cobre/análisis , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Matemática , Pseudomonas aeruginosa/crecimiento & desarrollo , EspectrofotometríaRESUMEN
The redox reaction between cytochrome c (Cyt c) (P-551) and the blue copper protein azurin, both from Pseudomonas aeruginosa, was studied using the temperature-jump technique. Two relaxation times were observed in a mechanism assumed to involve three equilibria. The fast relaxation time (0.4 less than tau less than 8 ms) was ascribed to the electron exchange step. The slow relaxation time (tau congruent to 37 ms) was assigned to a conformational equilibrium of the reduced azurin that was coupled through the electron exchange step to a faster conformational equilibrium of the oxidized Cyt c (P551). But because the Cyt c (P551) isomerization, being very rapid, was uncoupled from the two slower equilibria, and was assumed to involve no spectral change, the amplitude of its relaxation time (tau congruent to 0.1 ms) would be zero. At 25 degrees C and pH 7.0 the rate constants for the oxidation and reduction of Cyt c (P551) by azurin were 6.1 X 10(6) and 7.8 X 10(6) M-1 s-1, respectively; for the formation and disappearance of the reactive conformational isomer of azurin they were 12 and 17 s-1, respectively. The rates for the Cyt c (P551) isomerization could only be estimated at approximately 10(4) s-1. The thermodynamic parameters of each reaction step were evaluated from the amplitudes of the relaxations and from Eyring plots of the rate constants. Measurements of the overall equilibrium constant showed it to be temperature independent (5-35 degrees C), i.e. deltaHtot = 0. This zero enthalpy change was found to be compatible with the enthalpies calculated for the individual steps. In the electron exchange equilibrium, the values of the activation enthalpies were two to three times higher than the values published for various low molecular weight reagents in their electron exchange with copper proteins, yet the rate of exchange between Cyt c (P551) and azurin was some hundreds of times faster. This was explained in terms of the measured positive or zero entropies of activation that could result from a high level of specificity between the proteins particularly in areas of complementary charges. The mechanism of electron transfer was considered as essentially an outer sphere reaction, of which the rate could be approximated by the Marcus theory.