Inactivation, stabilization and redox regulation of iron-containing proteins

SummaryMicroperoxidases: kinetics and stability.Microperoxidases are small enzymes prepared by proteolytic digestion of cytochromes c. The proteolytic removal of most of the protein environment allows these enzymes to use a wide variety of substrates in peroxidase- and cytochrome- P-450-type reactions. A major drawback for the application of microperoxidases, however, is their rapid inactivation. The formation of high valent FeIV-oxo compounds, formed during catalysis, probably plays a key role in this inactivation. Chapter 2 describes the hydrogen peroxide-dependent para- hydroxylation of aniline by microperoxidases which have peptides attached to the herne moiety with different chain lengths (microperoxidases 6, 8 and 11, all prepared from horse-heart cytochrome c) and composition (microperoxidase 17, prepared from cytochrome c 550 from lhiobacillus versutus).Investigation of MP6, MP8 and MP11 (with peptide chains of 6, 8 and 11 amino acids, respectively) showed that these microperoxidases have similar V max values, but that the rate of inactivation is dependent on the length of the peptide chain. Inactivation rates (at 0.4 mM aniline) range from 0.141 s -1for MP6 to 0.091 s -1for M11. Although inactivation is rapid in all cases, a longer peptide chain apparently offers more protection against inactivation. The K m is also dependent on the length of the peptide chain, ranging from 0.82 mM for MP6 to 0.41 mM for MP11. In addition, the rates of inactivation for these microperoxidases decreases by about 50% when the aniline concentration is increased from 0.4 to 12 mM. This points at a role for the substrate in the inactivation process; possibly binding of aniline to the heme moiety also offers some protection.MP17 has kinetic properties that are not in fine with those of the microperoxidases described above: contrary to what is to be expected on the basis of the length of the attached peptide chain (17 amino acids) the rate of inactivation as well as the K m and V max values are higher than for the other microperoxidases. Increasing the aniline concentration from 0.4 to 12 mM only decreases the rate of inactivation of MP17 by about 10%. The observed correlation between V max and the rate of inactivation of the microperoxidases confirms the assumption that high valent heme compounds, formed during catalysis, are involved in inactivation. The different catalytic properties of MP17 must be caused by the specific properties of its peptide chain. MP17 does not bind a single peptide chain, as do the other inicroperoxidases, but two: a short chain (2 amino acids) and a longer chain (15 amino acids), which is the result of proteolytic cleavage between the heme-binding cysteines. Furthermore, the amino acid composition of the (broken) peptide chain is different from that of the other microperoxidases.The FeSII protein: stabilization of nitrogenase.The nitrogen fixing soil bacterium A. vinelandii produces a small iron-sulfur protein, known as the FeSII protein, that can bind to nitrogenase rendering it temporarily oxygen resistant. In chapter 3 of this thesis evidence is given that FeSII protein offers protection for the nitrogenase Fe protein from A. vinelandii against oxygen-inactivation by stabilizing the association of the Fe protein (Av2) with the MoFe protein (Av1).Av2 was exposed to oxygen (air) in the presence of MgADP under several conditions. Exposure of free Av2, i.e. without adding FeSII or Av1, to oxygen results in rapid inactivation: all activity is lost within 5 minutes. When Av1 is added in a 1 : 2 ratio with Av2 the inactivation is about 4 times slower. Under the experimental conditions approximately 75% of Av2 is associated with Av1, as Av1 [Av2(MgADP) 2 ] 2, at the start of the exposure to oxygen, which indicates that Av2 is protected against oxygen-inactivation by association with Av1 Addition of both Av1 and the FeSII protein results in a further decrease of the inactivation. When the FeSII protein, Av1 and Av2 are present in a 1 : 1 : 2 ratio, the rate of inactivation is 8 times slower, as compared to the sample containing only Av2 and Av1. This indicates that the FeSII protein stabilizes the interaction between Av2 and Av1. In the presence of MgADP and aluminum fluoride Av1 and Av2 form a stable Av1[Av2(AIF/MgADP) 2 ] 2 complex, from which Av2 can not dissociate. This nitrogenase complex proved to be the most stable of all investigated samples, which confirms the conclusion that the association of Av2 with Av1 protects Av2 against inactivation by oxygen.Evidence for the existence of two types of three-component protein complexes, formed by Av2, Av1 and the FeSII protein, was obtained from stopped-flow experiments. It was shown that when Av1 is present in a twofold excess with respect to the FeSII protein, the latter protein binds two Av1[Av2(MgADP) 2 ] 2 nitrogenase complexes, whereas when equimolar amounts of Av1 and FeSII are present in the incubation only one Av1[Av2(MgADP) 2 ] 2 nitrogenase complex is bound by the FeSII protein.Based on these experiments a model was set up to describe the reactions involved in the oxygen-inactivation and the reduction of the nitrogenase proteins under the various experimental conditions. Using this model and the kinetic parameters obtained from the various experiments, the data could be simulated best when 1: both the active and inactivated forms of Av2 were allowed to associate reversibly with Av1, 2: the rate constants for both interactions were identical and 3: the formation of both types of three-component complexes with the FeSII protein was taken into account. These simulations indicated that the formation of complexes between the FeSII protein and the Av1[Av2(MgADP) 2 ] 2 nitrogenase complex is an adequate explanation for the protection against oxidative inactivation of nitrogenase by the FeSII protein.The transition state complex of nitrogenase: redox regulation.Nitrogenase catalyzed reduction of N 2 to NH 3 requires the association of the nitrogenase Fe protein (Av2) with the nitrogenase MoFe protein (Av1), MgATP-dependent electron transfer from Av2 to Av1 and on-enzyme ATP hydrolysis followed by dissociation of Av2, with MgADP bound, from Av1. Aluminum fluoride and MgADP inhibit the dissociation of Av2 from Av1 by stabilizing the protein-protein complex of an intermediate of the on-enzyme MgATP hydrolysis reaction.Redox titrations and EPR spectroscopy revealed that the redox properties of FeMoco in Av1 remain unchanged in this complex, as compared to the purified, free MoFe protein. The redox properties of the [8Fe-7S] P-clusters in Av1 however, are markedly different. Upon oxidation, in a two-electron process, of the P-clusters in purified Av1 an EPR-signal appears with E m = -307 mV. In the AlF/MgADP stabilized complex this signal was not observed. Another signal, exhibited by the one electron oxidized P-clusters, appears at a potential lower than -500 mV (out of the range of the experiments). This signal disappears again at E m = -430 mV, which indicates that in the AlF/MgADP stabilized complex the abstraction of the second electron takes place at this potential. These observations indicate that the P-clusters in the AlF/MgADP stabilized complex have a different conformation, as compared to the P-clusters in free Av1.The [4Fe-4S] clusters in Av2 operate between the +1 and +2 oxidation levels. Upon reduction of the clusters in purified Av2 EPR-signals appear at E m = -473 mV when MgADP is bound to the protein or at E m = -440 mV when MgATP is bound. For the AlF/MgADP stabilized complex, however, no EPR-signals characteristic for reduced Av2 were observed and it was concluded that in the AlF/MgADP stabilized nitrogenase complex the midpoint redox potential of the [4Fe-4S] clusters in Av2 is lowered to less than -500 mV, out of the range of the experiments. This change in redox potential indicates that also the [4Fe-4S] clusters of Av2 in the AlF/MgADP stabilized complex have a different conformation, as compared to the clusters in free Av2.These results indicate that nitrogenase catalysis involves conformational redox regulation. Free Av2 has a relatively high redox potential which facilitates electron transfer to this protein from reductants (flavodoxin) in the cell. After binding of MgATP, Av2 changes its conformation. These changes in conformation lower the redox potential of the [4Fe-4S] cluster in Av2, thus inducing electron transfer to the P-clusters in Av1 The lowered redox potential of the P-clusters, resulting from conformational changes caused by binding of Av2 to Av1 facilitates electron transfer to FeMoco, the putative substrate-binding site, which has unchanged redox properties. The FeMoco clusters in turn transfer the electrons to the substrate, N 2 , to generate ammonia. After on-enzyme hydrolysis of ATP the Fe protein changes to the MgADP-bound conformation and dissociates from the MoFe protein to enter a new cycle of electron transfer.NifM: its role in the biosynthesis of the Fe protein.Chapter 5 reports on the investigation of the possible role of NifM in the maturation of the nitrogenase Fe protein from Azotobacter vinelandii. NifM was purified from E. coli harboring a recombinant plasmid that incorporated the nifM gene. The purified protein was colorless: the UV-visible spectrum gave no evidence for the presence of a cofactor. Comparison of the DNA and amino acid sequences of NifM with those of other proteins revealed that NifM has a relevant sequence homology to several proteins that are involved in protein folding / maturation, especially parvulin from E. coli. Parvulin is a peptidylprolyl cis-trans isomerase, or PPIase. PPIases catalyze the interconversion of the trans -isomer of proline into the cis - isomer. During protein synthesis, peptide bonds formed by the proline residues are in the trans conformation, but in folded proteins, appr. 15% of the residues have a cis conformation. In addition, the proper folding of a protein may require the temporary conversion of the trans conformation to the cis conformation. NifM could therefore be involved, as a PPIase, in the proper folding / maturation of the Fe apoprotein. PPIase activity of NifM could not be demonstrated. The role of NifM in the maturation of the Fe protein, however, was positively demonstrated: deletion of the nifM gene from A. vinelandii results in the production of inactive, precipitated Fe protein.