Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems.

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.

Comparing the rates and the activation parameters for the forward reaction between the triplet state of zinc cytochrome c and cupriplastocyanin and the back reaction between the zinc cytochrome c cation radical and cuproplastocyanin.

This is a comparative study of the photoinduced (so-called forward) electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I), between the triplet state of zinc cytochrome c (3Zncyt) and cupriplastocyanin [pc(II)], and the thermal (so-called back) electron-transfer reaction Zncyt+/pc(I) --> Zncyt/pc(II), between the cation (radical) of zinc cytochrome c (Zncyt+) and cuproplastocyanin [pc(I)], which follows it. Both reactions occur between associated (docked) reactants, and the respective unimolecular rate constants are kF and kB. Our previous studies showed that the forward reaction is gated by a rearrangement of the diprotein complex. Now we examine the back reaction and complare the two. We study the effects of temperature (in the range 273.3-302.9 K) and viscosity (in the range 1.00-17.4 cP) on the rate constants and determine enthalpies (DeltaH), entropies (DeltaS), and free energies (DeltaG) of activation. We compare wild-type spinach plastocyanin, the single mutants Tyr83Leu and Glu59Lys, and the double mutant Glu59Lys/Glu60Gln. The rate constant kB for wild-type spinach plastocyanin and its mutants markedly depends on viscosity, an indication that the back reaction is also gated. The activation parameters DeltaH and DeltaS show that the forward and back reactions have similar mechanisms, involving a rearrangement of the diprotein complex from the initial binding configuration to the reactive configuration. The rearrangements of the complexes 3Zncyt/pc(II) and Zncyt+/pc(I) that gate their respective reactions are similar but not identical. Since the back reaction of all plastocyanin variants is faster than the forward reaction, the difference in free energy between the docking and the reactive configuration is smaller for the back reaction than for the forward reaction. This difference is explained by the change in the electrostatic potential on the plastocyanin surface as Cu(II) is reduced to Cu(I). It is the smaller DeltaH that makes DeltaG smaller for the back reaction than for the forward reaction.

Effects of single and double mutations in plastocyanin on the rate constant and activation parameters for the rearrangement gating the electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin.

The unimolecular rate constant for the photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and spinach cupriplastocyanin is kF. We report the effects on kF of the following factors, all at pH 7.0: 12 single mutations on the plastocyanin surface (Leu12Asn, Leu12Glu, Leu12Lys, Asp42Asn, Asp42Lys, Glu43Asn, Glu59Gln, Glu59Lys, Glu60Gln, Glu60Lys, Gln88Glu, and Gln88Lys), the double mutation Glu59Lys/Glu60Gln, temperature (in the range 273.3-302.9 K), and solution viscosity (in the range 1. 00-116.0 cP) at 283.2 and 293.2 K. We also report the effects of the plastocyanin mutations on the association constant (Ka) and the corresponding free energy of association (DeltaGa) with zinc cytochrome c at 298.2 K. Dependence of kF on temperature yielded the activation parameters DeltaH, DeltaS, and DeltaG. Dependence of kF on solution viscosity yielded the protein friction and confirmed the DeltaG values determined from the temperature dependence. The aforementioned intracomplex reaction is not a simple electron-transfer reaction because donor-acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, deviate from the expectations based on precedents and because kF greatly depends on viscosity. This last dependence and the fact that certain mutations affect Ka but not kF are two lines of evidence against the mechanism in which the electron-transfer step is coupled with the faster, but thermodynamically unfavorable, rearrangement step. The electron-transfer reaction is gated by the slower, and thus rate determining, structural rearrangement of the diprotein complex; the rate constant kF corresponds to this rearrangement. Isokinetic correlation of DeltaH and DeltaS parameters and Coulombic energies of the various configurations of the Zncyt/pc(II) complex consistently show that the rearrangement is a facile configurational fluctuation of the associated proteins, qualitatively the same process regardless of the mutations in plastocyanin. Correlation of kF with the orientation of the cupriplastocyanin dipole moment indicates that the reactive configuration of the diprotein complex involves the area near the residue 59, between the upper acidic cluster and the hydrophobic patch. Kinetic effects and noneffects of plastocyanin mutations show that the rearrangement from the initial (docking) configuration, which involves both acidic clusters, to the reactive configuration does not involve the lower acidic cluster and the hydrophobic patch but involves the upper acidic cluster and the area near the residue 88.

Effects of viscosity and temperature on the kinetics of the electron-transfer reaction between the triplet state of zinc cytochrome c and cupriplastocyanin.

This is a study of the effects of viscosity (in the range of 0.8-790 cP), of temperature (in the range of 260.7-307.7 K), and of ionic strength (in the range of 2.5-20.0 mM) on the kinetics of photoinduced electron-transfer reaction 3Zncyt/pc(II) --> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and cupriplastocyanin at pH 7.0. The unimolecular rate constant is kF. The apparent activation parameters DeltaH*, DeltaS*, and DeltaG* for this reaction were obtained in experiments with aqueous glycerol solutions having a constant composition. The interpolation of kF values obtained at the constant composition into the dependence of kF on temperature at constant viscosity gave the proper activation parameters, which agree with those obtained in experiments with solutions having a constant viscosity. This agreement validates the latter method, which is more efficient than the former, for determining activation parameters of processes that are modulated by viscosity. The smooth change in kF is governed by the change in viscosity, not in other properties of the solvent, and it does not depend on the choice of the viscosigen. Donor/acceptor electronic coupling (HAB) and reorganizational energy (lambda), obtained by fitting of the temperature dependence of kF to the Marcus equation, are consistent with true electron transfer and with electron transfer that is coupled to, or gated by, a preceding structural rearrangement of the diprotein complex 3Zncyt/pc(II). The fact that at very high viscosity kF approaches zero shows that the reaction is probably gated throughout the investigated range of viscosity. Kinetic effects and noneffects of ionic strength, viscosity, and thermodynamic driving force indicate, but do not prove, that the reaction under consideration is gated. The kinetic effect of viscosity is analyzed in terms of two models. Because ln kF is a nonlinear function of ln eta, protein friction has to be considered in the analysis of viscosity effects on kinetics.

Effects of temperature on the kinetics of the gated electron-transfer reaction between zinc cytochrome c and plastocyanin. Analysis of configurational fluctuation of the diprotein complex.

This is a study of the effects of temperature (in the range 273.3-307.7 K) and of ionic strength (in the range 2.5-100 mM) on the kinetics of photoinduced electron-transfer reaction 3Zncyt/pc(II)--> Zncyt+/pc(I) within the electrostatic complex of zinc cytochrome c and cupriplastocyanin at pH 7.0. In order to separate direct and indirect effects of temperature on the rate constants, viscosity of the solutions was fixed, at different values, by additions of sucrose. The activation parameters for the reaction within the preformed complex, at the low ionic strength, are delta H++ = 13 +/- 2 kJ/mol and delta S++ = -97 +/- 4 J/K mol. The activation parameters for the reaction within the encounter complex, at the higher ionic strength, are delta H++ = 13 +/- 1 kJ/mol and delta S++ = -96 +/- 3 J/K mol. Evidently, the two complexes are the same. The proteins associate similarly in the persistent and the transient complex, i.e., at different ionic strengths. In both complexes, however, electron transfer is gated by a rearrangement, as previous studies from this laboratory showed. Changes in the solution viscosity modulate this rearrangement by affecting delta H++, not delta S++. The activation parameters are analyzed by empirical methods. The thermodynamic parameters delta H and delta S for the formation of the complex Zncyt/pc(II) are determined and related to changes in hydrophilic and hydrophobic surfaces upon protein association in three configurations. A difference between the values of delta H for the configuration providing optimal electronic coupling between the redox sites and the configuration providing optimal docking equals the experimental value delta H++ = 13 kJ/mol for the rearrangement of the latter configuration into the former. Enthalpy of activation may reflect a change in the character of the exposed surface as the diprotein complex rearranges. Entropy of activation may reflect tightening of the contact between the associated proteins.