Electron transport through the nitrogenase enzyme complex of Azotobacter vinelandii

A. Braaksma

Research output: Thesisinternal PhD, WU

Abstract

In Chapter VII (Discussion) the current thoughts as they were accepted in 1980 about the electron transport within the nitrogenase complex are summarized. The starting points and ideas about the subject of this thesis are also given. In this way, Chapter VII can be considered both as a summary and as a discussion for the scientific interested reader. For those, who are less familiar with this kind of research, the following summary gives the results of this study which emerge in the different chapters.<p/>In the biological N-cycle, the fixation of atmospheric dinitrogen is a key reaction. The enzyme nitrogenase catalyzes the reduction of dinitrogen to ammonia. The structure and function of nitrogenase isolated from different organisms is very similar, so most conclusions drawn from experiments with one particular nitrogenase can be extended to other nitrogenases as well. In this thesis experiments are described with nitrogenase from <u>Azotobacter</u><u>vinelandii</u> .<p/>The enzyme is <u>in</u><u>vitro</u> easily separated into two components, both essential for activity. The smallest component is called the Fe- protein and its function seems to be restricted to reduce specifically the bigger component, the MoFe-protein. This electron transfer only occurs when MgATP is present and MgATP is hydroylzed to MgADP and Pi during electron transfer. The actual reaction, the reduction of dinitrogen to ammonia, takes place on the MoFe-protein.<p/>Both proteins have iron/sulphur clusters, structures consisting of alternating iron and sulphide atoms, where the necessary electrons can be stored. The Fe-protein is thought to have only one iron/sulphur cluster of the [4Fe-4S] type, which is also found in ferredoxins. This kind of cluster can take up or donate only one electron.<p/>Although the experiments, which give rise to this hypothesis, seem very convincing, there are also observations described in literature which are not compatible with the view of one [4Fe-4S] cluster per molecule Fe-protein and a one electron donor/acceptor behaviour in redox reactions.<p/>This discrepancy is also present in this thesis. In chapter H for instance, redox reactions are described where the redox state of the Fe-protein is followed by means of EPR spectroscopy and by the activity of the intact enzyme complex. From the results obtained it can be concluded that possibly two electrons are involved in the redox reaction of Fe-protein with the MoFe protein. The experiments described in chapter III confirm this view. In these experiments the number of electrons Is determined which can be accepted by oxidized Fe-protein. In stopped-flow experiments dyeoxidized Fe-protein could accept two electrons from reduced methyl viologen. Since it is possible that the Fe-protein is over-oxidized by dyes, also the physiologically oxidized Fe-protein has been used. Also in this case the results obtained are more compatible with the view that Fe-protein behaves as a two electron donor/acceptor.<p/>Because of the fact that a [4Fe-4S] cluster can only react as a one electron redox centre, we examined our Fe-protein preparations by means of Proton Induced X-ray Emission Spectroscopy (PIXES) on the presence of other metal ions which could function as an electron acceptor. No other metals than Fe could be detected. But we found a much higher iron content than could be expected by assuming one [4Fe-4S] cluster. This gave rise to a thorough analysis of the iron/sulphide content of Fe-protein (chapter IV). The maximum number of iron and sulphide atoms found per molecule was eight. This combined with the results of Mössbauer experiments which indicate that all iron atoms are in an equal configuration, gave rise to the view that probably two [4Fe-4S] clusters are present in active Fe-protein.<p/>In chapter V, it was shown that the growth conditions determine the iron/sulphide content of isolated Fe-protein. During this investigation the maximum number of iron and sulphide atoms per molecule Fe-protein was six and not eight. The discrepancy between the values reported in chapters IV and V are discussed in chapter V.<p/>Mössbauer spectroscopy experiments were not restricted to the Fe-protein, but also the MoFe-protein has been subject of examination. The importance of our results are not only restricted to the fact that the specific activity of the protein sample used is much higher than that of others, but also the processing of the primary data has been altered. This resulted in a different ratio of Fe to Mo of the FeMo-cofactor of MoFe-protein. Due to this fact the composition of the other iron/sulphur clusters present in MoFe-protein is now questionable.<p/>It is clear that the work described in this thesis has not solved the problem of the composition and structure of the metal- sulphide clusters present in the nitrogenase proteins. Beside the described experiments which may lead to the discovery of the true nature of these clusters, an Important aspect is that the general accepted dogma that the structure of the metal-sulphide clusters of nitrogenase has been solved is questioned and reopened the reserach towards a better understanding of the structures and function of these clusters.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • Veeger, G., Promotor, External person
  • Haaker, H., Co-promotor, External person
Award date6 Nov 1985
Place of PublicationWageningen
Publisher
Publication statusPublished - 1985

Keywords

  • azotobacter
  • energy
  • nitrogenase
  • transfer

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