Electron Transport to Nitrogenase

Another assay which may be valuable and more specific than the chloroplast-nitrogenase assay is based on the observations of Massey and Hemmerich (1978) that light-activated 5-deazaflavin or deazariboflavin reduced electron carriers of negative redox potential. This system donated electrons to nitrogenase via azotobacter flavodoxin but not azotobacter ferredoxin I (Haaker and Veeger, 1977). 

The possibility that extra ferredoxins arise as artifacts of purification procedures cannot be discounted in some cases. However, there are clear differences of size, FeS content, redox potentials and in the effect of KsFeCNg on the epr spectra of the two ferredoxins fromM.flavum (Yates et al., 1978) and also in the size, FeS content, physical location and physiological condi- 

Organism Ferredoxins Mo- Types lecu- of lar FeS Em weight cluster (mV) Functional oxidation Ref-levels erence  

The epr signal of oxidized A. vinelandii ferredoxin I integrates to two electrons/mol (Sweeney et al., 1975) whereas that of ferredoxin I from M. ftavum integrates to a maximum of only 0.35 electrons/mol (Yates et al., 1978). This may mean that the two 4Fe4S clusters of M. flavum ferredoxin are sufficiently close to interact to diminish the epr signal (cf. nitrogenase Fe protein. Section II,D,5) whereas those of the A. vinelandii ferredoxin are remote. 

Flavodoxins are small flavoproteins characterized by a single FMN group per molecule. They have three oxidation states, fully oxidized, semiquinone and fully reduced or hydroquinone, each of which has a characteristic uv-visible spectra (Fig. 8). The fully oxidized form is yellow, the semiquinone intensely blue and the hydroquinone is pale yellow to colorless. Only the 

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Fig. 10. Scheme for electron transport to nitrogenase in A. vinelandii involving an energized membrane (boxes) (from Haaker and Veeger, 1977). 

Peschek GA and Schmetterer G (1982) Evidence for plastoquinol-cytochrome f/b-563 reductase as a common electron donor to P700 and cytochrome oxidase in cyanobacteria, Biochem. Biophys. Res. Commun. 108, 1188-1195. Schrautemeier B, Bohme H and Boger P (1983) In vitro studies on pathways and regulation of electron transport to nitrogenase with a cell-free extract from heterocysts of Anabaena variabilis. Arch. Microbiol., submitted. 

II. ELECTRON TRANSPORT TO NITROGENASE IN HETEROCYSTS OF ANABAENA VARIABILIS... 

Vallejos 1983 and ref. 3 therein) would greatly favour this regulatory mechanism. Energized membranes are not necessary for regulation of electron transport to nitrogenase in heterocysts (cf. Haaker et al. 1980 Hawkesford et al. 1981). Glycolysis rather than the oxidative pentose-phosphate pathway is likely to supply electrons for nitrogen fixation in heterocysts. 

Schrautemeier B, Bdhme H and Bdger P (1983) In vitro studies on pathways and regulation of electron transport to nitrogenase with a cell-free extract from heterocysts of Anabaena variabilis. Arch. Microbiol., in press. 

A high NADPH/NADP ratio generated by photoreduction of NADP with NADH (or H2) is a prerequisite for photosynthetic electron transport to nitro-genase. The NADPH/NADP ratio may balance cyclic and non-cyclic electron flow around PS I, adjusting the optimum ATP/e-ratio for nitrogenase (or other biosynthetic activities) to operate. A fixed spatial arrangement of FNR between ferredoxin and the b /f-complex (Fig.4 see Carrillo,... 

As noted earlier, nitrogenase is made up of two proteins, the iron protein, and the molybdenum-iron protein, and will be linked to an electron-transport chain. The iron protein accepts electrons from this chain (a ferredoxin or flavodoxin in vivo, or dithionite in vitro) and transfers them to the molybdenum-iron protein. The MoFe protein is then able to reduce a number of substrates in addition to dinitrogen. No replacement electron donor will function instead of the iron protein. 

Nitrogenase catalyzes the ATP-driven eight-electron and eight-proton reduction of N2 to 2 NH3 and H2 and offers the most complex and fascinating example of Fe-S cluster mediated electron transfer. The electron transport chain involves transfer of electrons from the subunit-bridging [4Fe-4S] center on the homodimeric Fe-protein, to the subunit-bridging double-cubane [8Fe-7S] P-cluster (see... 

The smaller protein has a molecular mass of 60000-70000 daltons. Extended X-ray absorption fine structure (EXAFS) studies show that it includes a single Fe4S4 cubane cluster (P-cluster) similar to that in ferredoxins, which are important for various electron transport chains in living organisms. The smaller protein in the nitrogenase system is a strongly reductive reductase that is obviously responsible for the transfer of electrons to the larger protein. 

Fig. 6. Current concepts of bacteroid metabolism relating to N2 fixation and ammonia production in legume root nodules. The relationships to nitrogenase activity of carbohydrate utilization, electron transport, and H2 cycling, are idicated. Nodules without an uptake hydro-genase lose energy through Hj evolution. Leghemoglobin, LHb flavodoxin hydroquinone, FldHj flavodoxin semiquinone, FldH. (Based on Evans et al. (1979) and reproduced with permission.)...

The two components of the Fe-Mo protein nitrogenase, which is responsible for the fixation of nitrogen by microbes, have been purified and characterized from several sources. It seems that the enzyme acts through an elaborate mechanism which involves electron transport from one protein to the other promoted by reaction with Mg-ATP. Further results with a nitrogenase model have been reported. 

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