SUMMARY: H2 production by nitrogenase was investigated in O2-, N2-, C- or SO−24-limited continuous cultures of Azotobacter chroococcum. H2 evolution occurred under air and was augmented when Ar replaced N2. Pretreatment of each culture with 40% acetylene in air or Ar/O2 mixtures inhibited the H2-uptake hydrogenase and increased H2 evolution. H2 production in each culture was O2-dependent, and, like acetylene reduction by nitrogenase, it increased to a maximum at an optimum O2 concentration and was inhibited by excess O2. The molar ratio of H2 produced to N2 reduced was least at the optimum O2 concentration and increased sharply under O2-limiting or O2-inhibiting conditions in vivo. The minimum value achieved was approximately 1 in O2-, N2- or C-limited cultures, or with purified nitrogenase components assayed in vitro, and 0·5 in SO42−-limited cultures. These differences may reflect different mechanisms of N2 reduction in vivo. Estimates of the levels of nitrogenase component proteins indicated Ac1: Ac2 ratios of 1 or less. However, Ac2 activity may be inhibited under O2-limiting or O2-inhibiting conditions. The maximum ratio of the number of electrons transferred to nitrogenase and to O2 was 0·1 in assays with O2-, C- or SO42−-limited cultures; thus, recycling by the H2-uptake hydrogenase of H2 produced by nitrogenase could contribute up to 7% of the total energy produced by respiration.
AndersenK.,
ShanmugamK.T.1977; Energetics of biological nitrogen fixation: determination of the ratio of formation of H2 to NH4+catalysed by nitrogenase of Klebsiella pneumoniae in vivo. Journal of General Microbiology 103:107–122
BulenW.A.1976; Nitrogenase from Azotobacter vinelandii and reactions affecting mechanistic interpretations. In Proceedings of the First International Symposium on Nitrogen Fixation1 pp. 177–195NewtonW.E.,
NymansC.
Edited by Pullman, Washington:: Washington State University Press.;
ChattJ.1980; Chemistry relevant to the biological fixation of nitrogen. In Nitrogen Fixation Annual Proceedings of the Phytochemical Society of Europe 18 pp. 1–18StewartW.D.P.,
GallonJ.R.
Edited by London:: Academic Press.;
EvansH.J.,
Ruiz-ArgÜesoT.,
JenningsN.,
HanusJ.1977; Energy coupling efficiency of symbiotic nitrogen fixation. In Genetic Engineering for Nitrogen Fixation pp. 333–354HollaenderA.
Edited by New York:: Plenum Press.;
HochG.E.,
SchneiderK.C.,
BurrisR.H.1960; Hydrogen evolution and exchange and conversion of NzO to N2 by soybean root nodules. Biochimica et biophysica acta 37:273–279
KennedyC.,
EadyR.R.,
KondorosiE.,
RekoshD.K.1976; The molybdenum-iron protein of Klebsiella pneumoniae nitrogenase: evidence for non-identical subunits from peptide ‘mapping’. Biochemical Journal 155:383–389
MaryanP.S.,
VorleyW.T.1979; An improved spectrophotometric method for the determination of ammonia with particular relevance to in vitro nitrogenase activity. Laboratory Practice 28:251–252
NewtonW.E.,
BulenW.A.,
HadfieldK.L.,
StiefelE.I.,
WattG.D.1977; HD formation as a probe for intermediates in N2 reduction. In Recent Developments in Nitrogen Fixation pp. 119–130NewtonW.E.,
PostgateJ.R.,
Rodriguez-BarruecoC.
Edited by London:: Academic Press.;
PartridgeC.D.P.,
WalkerC.C.,
YatesM.G.,
PostgateJ.R.1980; The relationship between hydrogenase and nitrogenase in Azotobacter chroococcum: effect of nitrogen sources on hydrogenase activity. Journal of General Microbiology 119:313–319
PostgateJ.R.1974; Prerequisites for biological nitrogen fixation in free-living heterotrophic bacteria. In The Biology of Nitrogen Fixation pp. 663–686QuispelA.
Edited by Amsterdam:: North Holland.;
ScheringsG.,
HaakerH.,
VeegerC.1977; Regulation of nitrogen fixation by Fe-S protein II in Azotobacter vinelandii. European Journal of Biochemistry 77:1207–1211
SchrauzerG.N.1977; Nitrogenase model systems and the mechanism of biological nitrogen reduction: advances since 1974. In Recent Developments in Nitrogen Fixation pp. 109–118NewtonW.,
PostgateJ.R,
Rodriguez-BarruecoC.
Edited by London:: Academic Press.;
SchubertK.R.,
EvansH.J.1976; Hydrogen evolution: a major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proceedings of the National Academy of Sciences of the United States of America 73:1207–1211
ShahV.K.,
DavisL.C.,
BrillW.J.1975; Nitrogenase. VI. Acetylene reduction assay: dependence of nitrogen fixation estimates on component ratio and acetylene concentration. Biochimica et biophysica acta 384:353–359
SilversteinR.,
BulenW.A.1970; Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions. Biochemistry 9:3809–3815
ThorneleyR.N.F.,
EadyR.R.1977; Nitrogenase of Klebsiella pneumoniae: distinction between proton-reducing and acetylene-reducing forms of the enzyme. Biochemical Journal 167:457–461
ThorneleyR.N.F.,
EadyR.R.,
LoweD.J.1978; Biological nitrogen fixation by way of an enzyme-bound dinitrogen-hydride intermediate. Nature; London: 272557–558
YatesM.G.,
PlanquÉK.1975; Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins. European Journal of Biochemistry 60:467–476