Temperature and pH dependence of the proton magnetic hyperfine resonances of cytochrome c peroxidase-cyanide. Evidence for hindered vinyl group rotation as a mediator of the enzymes activity

Proton nmr studies of the hyperfine resonances of cytochrome c peroxidase reveal that two pH-dependent processes can be monitored. One of these is the simple pH titration of a resonance which has been previously assigned to the alpha-vinyl proton at heme position 4. Combined with this proton's temperature dependence, the pH data indicate that the rotational position of vinyl 4 is changing with a pK which is similar to that which regulates the enzyme's activity. The second process, slow on the nmr time scale, occurs above pH 8. This is beyond what is normally considered to be the optimum pH range for cytochrome c peroxidase's activity and we interpret this to indicate a protein conformational change.     

Electrostatic and steric control of electron self-exchange in cytochromes c, c551, and b5.

The ionic strength dependence of the electron self-exchange rate constants of cytochromes c, c551, and b5 has been analyzed in terms of a monopole-dipole formalism (van Leeuwen, J.W. 1983. Biochim. Biophys. Acta. 743:408-421). The dipole moments of the reduced and oxidized forms of Ps. aeruginosa cytochrome c551 are 190 and 210 D, respectively (calculated from the crystal structure). The projections of these on the vector from the center of mass through the exposed heme edge are 120 and 150 D. For cytochrome b5, the dipole moments calculated from the crystal structure are 500 and 460 D for the reduced and oxidized protein; the projections of these dipole moments through the exposed heme edge are -330 and -280 D. A fit of the ionic strength dependence of the electron self-exchange rate constants gives -280 (reduced) and -250 (oxidized) D for the center of mass to heme edge vector. The self-exchange rate constants extrapolated to infinite ionic strength of cytochrome c, c551, and b5 are 5.1 x 10(5), 2 x 10(7), and 3.7 x 10(5) M-1 s-1, respectively. The extension of the monopole-dipole approach to other cytochrome-cytochrome electron transfer reactions is discussed. The control of electron transfer by the size and shape of the protein is investigated using a model which accounts for the distance of the heme from each of the surface atoms of the protein. These calculations indicate that the difference between the electrostatically corrected self-exchange rate constants of cytochromes c and c551 is due only in part to the different sizes and heme exposures of the two proteins.     

Spectroscopic and oxidation-reduction properties of Rhodobacter capsulatus cytochrome c1 and its M183K and M183H variants

Two variants of the cytochrome c1 component of the Rhodobacter capsulatus cytochrome bc1 complex, in which Met183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type cytochrome c1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation-reduction titrations of the heme in purified cytochrome c1 revealed multiple redox forms. Titrations of the purified cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of cytochrome c1 in the intact bc1 complex and in a subcomplex missing the Rieske iron-sulfur protein were fully reversible. An Em7 value of -330 mV was measured for the single disulfide bond in cytochrome c1. The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of cytochrome c1.     

1H NMR structural characterization of the cytochrome c modifications in a micellar environment

The interaction of cytochrome c with micelles of sodium dodecyl sulfate was studied by proton NMR spectroscopy. The protein/micelles ratio was found to be crucial in controlling the extent of the conformational changes in the heme crevice. Over a range of ratios between 1:30 and 1:60, the NMR spectra of the ferric form display no paramagnetic signals due to a moderately fast exchange between intermediate species on the NMR time scale. This is consistent with an interconversion of bis-histidine derivatives (His18-Fe-His26 and His18-Fe-His33). Further addition of micelles induces a high-spin species that is proposed to involve pentacoordinated iron. The resulting free binding site, also encountered in the ferrous form, is used to complex exogenous ligands such as cyanide or carbon monoxide. Attribution of the heme methyls was performed by means of exchange spectroscopy through ligand exchange or electron transfer. The heme methyl shift pattern of the micellar cyanocytochrome in the ferric low spin form is different from the pattern of both the native and the cyanide cytochrome c adduct, in the absence of micelles, reflecting a complete change of the heme electronic structure. Analysis of the electron self-exchange reaction between the two redox states of the micellar cyanocytochrome c yields a rate constant of 2.4 x 10(4) M(-1) s(-1) at 298 K, which is surprisingly close to the value observed in the native protein.     

EPR study of the redox interactions in cytochrome c3 from Desulfovibrio vulgaris Miyazaki

We present a new examination of the EPR redox titration data for the tetraheme cytochrome c3 from Desulfovibrio vulgaris Miyazaki. Our analysis includes the contribution of the interaction potentials between the four redox sites and is based on the model previously developed for the study of cytochrome c3 from Desulfovibrio desulfuricans Norway. We observed, as for D. desulfuricans Norway cytochrome c3, that the conformation of the heme with the lowest redox potential, heme 4, is sensitive to the redox state of the heme with the highest potential, heme 1. However in D. vulgaris Miyazaki cytochrome c3 spectral simulations show that heme 4 is present in two conformational states which interconvert partially when heme 1 is reduced. The sets of redox parameters which satisfy the fitting procedure of the titration curves are in the following domain: -250 mV less than or equal to e41 less than or equal to -220 mV, -325 mV less than or equal to e2 less than or equal to -320 mV, -335 mV less than or equal to e3 less than or equal to -330 mV, -360 mV less than or equal to e4 less than or equal to -355 mV, -5 mV less than or equal to I12 less than or equal to 20 mV, -10 mV less than or equal to I13 less than or equal to 5 mV, -15 mV less than or equal to I23 less than or equal to -5 mV, -15 mV less than or equal to I24 less than or equal to -10 mV, -25 mV, less than or equal to I34 less than or equal to -15 mV. As in D. desulfuricans Norway cytochrome c3 the interactions are moderate. Simple electrostatic considerations suggest that these moderate values could be related to the large accessibility of the hemes to the solvent. Our work does not confirm the existence of a cooperative interaction between heme 2 and heme 3 which has been proposed on the basis of electrochemical measurements.     

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2-

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2- have been made. The reaction was found to follow a second-order rate law: k 4.2 x 10(3) M(-1) s(-1) [25 degrees, micro0.1 M, pH 7.0 (phosphate)]; deltaH+/+ 3.2 kcal/ mol; AS+/+ -30 cal/mol-deg. The electrostatics-corrected self-exchange rate constant (k11 corr) calculated for cytochrome c551 based on the Fe(EDTA)2- cross reaction is 2 M(-1) s(-1), as compared to a value of 6 M(-1) s(-1) for horse heart cytochrome c. The close correspondence of the two k11 corr values is taken as an indication that the two proteins employ very similar electron transfer mechanisms in their reactions with Fe(EDTA)(2-). It is proposed that this mechanism involves reagent contact, but little protein conformational change, at the partially exposed heme edge.     

EPR determination of interaction redox potentials in a multiheme cytochrome: cytochrome c3 from Desulfovibrio desulfuricans Norway

In cytochromes c3 which contain four hemes per molecule, the redox properties of each heme may depend upon the redox state of the others. This effect can be described in terms of interaction redox potentials between the hemes and must be taken into account in the characterization of the redox properties of the molecule. We present here a method of measurement of these interactions based on the EPR study of the redox equilibria of the protein. The microscopic and macroscopic midpoint potentials and the interaction potentials are deduced from the analysis of the redox titration curves of the intensity and the amplitude of the EPR spectrum. This analysis includes a precise simulation of the spectrum of the protein in the oxidized state in order to determine the relative contribution of each heme to the spectral amplitude. Using our method on cytochrome c3 from D. desulfuricans Norway, we found evidence for the existence of weak interaction potentials between the hemes. The three interaction potentials which have been measured are characterized by absolute values lower than 20 mV in contrast with the values larger than 40-50 mV which have been reported for cytochrome c3 from D. gigas. Simulations of the spectra of samples poised at different potentials indicate a structural modification of the heme with the most negative potential during the first step of reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration and the hemes in the tridimensional structure is discussed.     

Active site structure and dynamics of cytochrome c3 from Desulfovibrio gigas immobilized on electrodes

Cytochrome c3 from Desulfovibrio gigas is electrostatically adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid. The redox equilibria and electron transfer dynamics of the adsorbed four-heme protein are studied by surface enhanced resonance Raman spectroscopy. Immobilization on the coated electrodes does not cause any structural changes in the redox sites. The potential-dependent stationary experiments distinguish the redox potential of heme IV (-0.19 V versus normal hydrogen electrode) from those of the other hemes for which an average value of -0.3 V is determined. Taking into account the interfacial potential drops, these values are in good agreement with the redox potentials of the protein in solution. The heterogenous electron transfer between the electrode and heme IV of the adsorbed cytochrome c3 is analyzed on the basis of time-resolved experiments, leading to a formal electron transfer rate constant of 15 s(-1), which is a factor of 3 smaller than that of the monoheme protein cytochrome c.     

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.     

Chromatium flavocytochrome c: kinetics of reduction of the heme subunit, and the flavocytochrome c-mitochondrial cytochrome c complex

The kinetics of reduction of Chromatium vinosum flavocytochrome c heme subunit by exogenous flavin neutral semiquinones generated by laser flash photolysis have been investigated. Unlike the holoprotein, the isolated heme subunit was appreciably reactive with lumiflavin neutral semiquinone. The measured rate constant for the reaction (2.7 X 10(7) M-1 S-1) was comparable to those of c-type cytochromes having similar redox potentials. The ionic strength dependence of the reaction with FMN neutral radical indicated that the heme subunit had a small negative charge at the site of reduction. Taken together, these results suggest that the active site of the heme subunit is buried on complexation with the flavin subunit in the holoprotein. Horse cytochrome c formed a strong complex with Chromatium, but not Chlorobium, flavocytochrome c. Possible physiological electron acceptors such as HiPIP, cytochrome c', and cytochrome c-555 apparently did not bind to the flavocytochromes c. The rate constant for reduction by lumiflavin radical of horse cytochrome c complexed to flavocytochrome c was about twofold smaller than for reduction of horse cytochrome c alone. Flavocytochrome c was itself unreactive with exogenous flavin semiquinones. The ionic strength dependence of the reduction of the complex by FMN radical was also smaller than for horse cytochrome c in the absence of flavocytochrome c. Sulfite, which forms an adduct with the protein-bound FAD (FAD is bound in an 8-alpha-S-cysteinyl linkage), did not affect the reduction of horse cytochrome c in its complex with flavocytochrome c. We conclude that horse cytochrome c is reduced directly by exogenous flavins in its complex with flavocytochrome c, although the kinetics are slightly modified. These results are not unlike observations made with complexes of mitochondrial cytochrome c with cytochrome oxidase or cytochrome b5.     

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.     

Kinetics of inter- and intramolecular electron transfer of Pseudomonas nautica cytochrome cd 1 nitrite reductase: regulation of the NO-bound end product

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.     

Assignment of individual heme EPR signals of Desulfovibrio baculatus (strain 9974) tetraheme cytochrome c3. A redox equilibria study

An EPR redox titration was performed on the tetraheme cytochrome c3 isolated from Desulfovibrio baculatus (strain 9974), a sulfate-reducer. Using spectral differences at different poised redox states of the protein, it was possible to individualize the EPR g-values of each of the four hemes and also to determine the mid-point redox potentials of each individual heme: heme 4 (-70 mV) at gmax = 2.93, gmed = 2.26 and gmin = 1.51; heme 3 (-280 mV) at gmax = 3.41; heme 2 (-300 mV) at gmax = 3.05, gmed = 2.24 and gmin = 1.34; and heme 1 (-355 mV) at gmx = 3.18. A previously described multi-redox equilibria model used for the interpretation of NMR data of D. gigas cytochrome c3 [Santos, H., Moura, J.J.G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283-296] is discussed in terms of the EPR results.     

Ferricytochrome c oxidation of cobaltocytochrome c. Comparison of experiments with electron-transfer theories.

Electron transfer from cobaltocytochrome c to ferricytochrome c has been studied by stopped-flow kinetics. The second-order rate constant at pH 7.0, 0.1 ionic strenght, 0.2 M phosphate, and 25 degrees C is 8.3 x 103 M-1 s-1. The activation parameters obtained from measurements made between 20 and 50 degrees C are deltaHnot equal to = 2.3 kcal mol-1 and deltaSnot equal to = -33 eu. The rate constant is not significantly dependent on ionic strength; it is also relatively independent of pH between the pK values for conformation transitions. The rate diminishes at pH greater than 12. The self-exchange reaction of cobalt cytochrome c was investigated with pulsed Fourier transform 1H NMR. The rate is too slow on the 1H NMR scale; it is estimated to be less than 133 M-1 s-1. These results together with the self-exchange rates of iron cytochrome c [Gupta, R.K., Koenig, S. H., and Redfield, A. G. (1972), J. Magn. Reson. 7, 66] were analyzed by theories of Jortner and Hopfield. The theories predict the self-exchange of Cocyt c to be too slow for 1H NMR determination. The rate constant calculated by the nonadiabatic multiphonon electron-tunneling theory for the Fecyt c-Fecyt c+ and Cocyt c-Fecyt c+ electron transfers are in good agreement with experiments.     

Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.     

Proton NMR spectroscopy of cytochrome c-554 from Alcaligenes faecalis

Cytochrome c-554 from the bacterium Alcaligenes faecalis (ATCC 8750) is a respiratory electron-transport protein homologous to other members of the cytochrome c family. Its structure has been studied by 1H NMR spectroscopy in both the ferric and ferrous states. The ferric spectrum is characterized by downfield hyperfine-shifted heme methyl resonances at 46.25, 43.60, 38.40, and 36.73 ppm (25 degrees C, pH 7.1). Chemical shifts of these resonances change with temperature opposite to expectations derived from Curie's law. The pH behavior of the hyperfine-shifted resonances titrates with a pK of 6.3 that has been interpreted as due to ionization of a heme propionate. In the ferrous state, heme methyl, meso, and thioether bridge resonances have been observed and assigned. All aromatic proteins have been assigned according to the side chain of origin, and the structural environment about the sole tryptophan residue has been examined. The electron-transfer rate between ferric and ferrous forms has been estimated to be on the order of 3 X 10(8) M-1 s-1, which is the largest such self-exchange rate yet observed for a cytochrome.     

Thermodynamics of carbon monoxide binding to monomeric cytochrome c'

The thermodynamic parameters for carbon binding to monomeric Rhodopseudomonas palustris cytochrome c' are determined. An enthalpy change for CO(aq) binding to the cytochrome is measured directly by titration calorimetry as -6.7 +/- 0.2 kcal/mol of heme, the CO binding equilibrium constant is measured at 35 degrees C as (1.96 +/- 0.05) X 10(5) M-1, and the binding equilibrium constant at 25 degrees C is calculated from the van't Hoff equation as (2.8 +/- 0.1) X 10(5) M-1. Comparison of the results to the known energetics of CO binding to dimeric cytochrome c', where the CO binding site is buried in the protein interior, indicates that the heme binding site on the monomer form is, in contrast, more exposed.     

Magnetic studies on the ferri-haem undecapeptide of cytochrome c.

The pH-dependence of the magnetic moment of a ferri-haem undecapeptide, produced by peptic digestion of cytochrome c, has been measured in aqueous solution using a nuclear magnetic resonance method. Below pH 3 the magnetic moment is consistent with the presence of hydroxo-bridged dimers of high-spin iron(III). Above pH 7 the iron(III) is low-spin, the spin crossover conforming to a simple titration curve with pK 6.3 (n=1). This transition is discussed with reference to spectrophotometric studies of ligand binding at the haem.