Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide. Electrostatic and redox potential effects

We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's). The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory. In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller. The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions. An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this. The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic. The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster. For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced. The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions. Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions. This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of the redox potential on the activity of Clostridium pasteurianum and Chromatium hydrogenases

The oxidation of reduced methyl viologen by Clostridium pasteurianum or Chromatium hydrogenases as a function of the redox potential of the reaction mixture has been studied spectrophotometrically. The same results were obtained effecting the reduction of methyl viologen either with dithionite or with metallic zinc. With C. pasteurianum hydrogenase a gaussian pattern was obtained. This is indicative of a process involving two one-electron steps, which suggests that [4Fe-4S]²⁺ is the catalytically active species. On the contrary, in the case of Chromatium hydrogenase the data follow a sigmoidal pattern corresponding to a two-electron reduction process, which demonstrates that the redox site must be totally reduced to be active. This finding is at variance with the previously reported electron paramagnetic resonance spectra, which suggest that the single [4Fe-4S] cluster of this enzyme transfers or accepts only one electron.     

The immunological properties of the Clostridium pasteurianum rubredoxin

An antibody to Clostridium pasteurianum rubredoxin was found in goat serum after multiple injections of the protein. This antibody was purified by adsorption and elution from a Sepharose-rubredoxin column. The purified antibody formed a precipitating complex with C. pasteurianum rubredoxin and aporubredoxin, but not with the rubredoxin from Micrococcus aerogenes, Peptostreptococcus elsdenii, and Pseudomonas oleovorans. All rubredoxins tested were adsorbed to Sepharose-antirubredoxin columns indicating that each could form a soluble complex with anti-C. pasteurianum rubredoxin. The relative affinity of the antirubredoxin for the various rubredoxins was demonstrated by its ability to inhibit the rubredoxin-dependent reduction of cytochrome c by NADPH in the presence of NADP-ferredoxin oxidoreductase. These data suggest that there are two antigenic determinants in C. pasteurianum rubredoxin and only one such determinant in the rubredoxin from other organisms which are recognized by anti-C. pasteurianum rubredoxin.     

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.     

Redox and spectral properties of flavodoxin from Anabaena 7120

We report here on the spectrophotometric and electrochemical properties of the flavodoxin from Anabaena 7120 and compare these properties with those of flavodoxins that have been studied previously. Molar absorption coefficients have been determined for all three oxidation states of this protein, at various wavelengths. For oxidized flavodoxin, molar absorption coefficients for the absorption maxima at 464 and 373 nm were 9200 and 8500 M-1 cm-1, respectively. Reduction by the first electron produced a neutral blue semiquinone which exhibited an absorption maximum at 575 nm. The molar absorption coefficients at 575 nm were 200 M-1 cm-1 for the oxidized form, 5100 M-1 cm-1 for the semiquinone form, and 250 M-1 cm-1 for the hydroquinone form. Redox potentials have been determined, in the pH range of 6.0 to 8.5, for both electron transfers. At pH 7.0, the midpoint potential values for the first and second electron transfers were -0.196 and -0.425 V, respectively. We determined that the first electron transfer is pH dependent and that a proton transfer accompanies this one electron transfer. It was also determined that the second electron transfer is pH independent in the pH range of 6.0 to 8.5.     

Regulation and properties of an invertase from Clostridium pasteurianum.

An intracellular invertase was induced in cultures of Clostridium pasteurianum utilizing sucrose as its carbon source for growth. This enzyme synthesis could be repressed by the addition of fructose of a sucrose-growing culture. In contrast, invertase activity was not affected by the addition of glucose to sucrose-growing cells and this enzyme could be induced in a glucose-metabolizing culture by the addition of sucrose. This enzyme was purified 10.5-fold over the induced lese, EC 3.2.1.26) by substrate-specificity studies. Invertase had a pH optimum of 6.5 and an apparent Km of 79.5 mM for sucrose, and required high concentration of potassium phosphate for maximum activity. Invertase was completely inactivated by a 2-min heat treatment at 60 degrees C. This enzyme was strongly inhibited by p-hydroxymercuribenzoate (pCMB) and weakly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), while cysteine could substantially reverse pCMB) inhibition, suggesting that sulfhydryl group(s) were necessary for invertase activity.     

Oxidation reduction properties of nitrogenase from Clostridium pasteurianum W5

Dithionite reduced azoferredoxin and molybdoferredoxin from $\text{Clostridium pasteurianum}$ W5 were oxidatively titrated with various electron acceptors. The AzoFd gave up 0.87 electrons per AzoFd monomer (27,500 mol. wt.). The oxidation reduction potential of AzoFd, determined by equilibrium with redox dyes, was ?0.240 V. Dithionite reduced MoFd gave up 3.6 electrons per MoFd tetramer (220,000 mol. wt.). The oxidation reduction potential for MoFd was ?0.070 V. Because the potential of $\text{this}$ MoFd half cell is so positive, the electrons removed during this oxidation may not be those that reduce dinitrogen.     

Purification and properties of paramagnetic protein from Clostridium pasteurianum W5.

The purification to homogeneity of the non-heme iron protein, sometimes referred to as either "red protein" or "paramagnetic protein", from Clostridium pasteurianum W5 extracts is described and its physicochemical properties studied. This paramagnetic protein (g= 1.94) has a molecular weight of about 25000 and contains two iron and two acid-labile sulfur atoms per mol of protein. Its midpoint potential at pH 7.5, as determined by electron paramagnetic resonance titration, is -300 mV. Optical circular dichroism and electron paramagnetic resonance spectra of the paramagnetic protein are similar to those of two iron-two acid-labile sulfur ferredoxins. The biochemical reduction of the purified protein was also studied.