Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex

Abstract

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2v) to the VFe protein (Ac IV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23°C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145,[391][392][393][394][395][396]. Extrapolation of the dependence of kobs on [MgATP] [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex. INTRODUCTIONThe recently discovered vanadium-containing nitrogenases from Azotobacter chroococcum (Robson et al., 1986;Eady et al., 1987Eady et al., , 1988a and Azotobacter vinelandii (Hales et al., 1986a,b) catalyse the reduction of N2 to NH3 in a reaction coupled to the hydrolysis of MgATP and the evolution of H2. Although a detailed mechanism for the action of the intensively studied molybdenum-containing nitrogenase from Klebsiella pneumoniae has been proposed [see Thorneley & Lowe (1985) and Orme-Johnson (1985) for reviews], kinetic data for the purified vanadium nitrogenase have only been obtained under steady-state assay conditions with necessarily limited mechanistic conclusions (Hales et al., 1986a,b;Eady et al., 1987Eady et al., , 1988aDilworth et al., 1987Dilworth et al., , 1988.The vanadium nitrogenase systems in A. chroococcum and A. vinelandii are genetically distinct from the molybdenum nitrogenase systems that are normally expressed in these organisms when routinely grown on nitrogen-free medium containing molybdenum (Robson et al., 1986;Bishop et al., 1986;Hales et al., 1986a . It has been shown that, unlike the MoFe proteins, which have an a2fi2 subunit structure, Aclv has an additional subunit of M; 14000 and an oc2fl282 subunit structure (Eady et al., 1988b). The distribution of these metal and sulphide ions between the two types of subunit (M 50000 and 55000, analogous to those of MoFe proteins) is not known, although an iron-and vanadium-containing cofactor (FeVaco), analogous to the iron-and molybdenum-containing cofactor (FeMoco) of the MoFe proteins, has been isolated (Smith et al., 1988). The oxidation level and ligand environment of the vanadium in the protein has been shown by V K-edge X-rayabsorption spectroscopy (Arber et al., 1987) Hales et al. (1986b).The vanadium nitrogenase from A. chroococcum, like the molybdenum nitrogenases, requires MgATP and a source of low-potential electrons (Na2S204 in vitro) to reduce substrates such as H+, N2 and C2H2. Steady-state assays with Ac 1V or Ac 1 Mo in combination with Ac2v or Ac2MO showed that, although the heterologous nitrogenases (Ac 1VAc2M0 and Ac 1MoAc2v) are active, it is the Ac V or AcIMo that determines substrate specificity. AclV with either Ac2V or Ac2MO evolves significantly more H2 concomitant with C2H2 reduction than does AclM0 with Ac2M`or Ac2v (Eady et al., 1987). Aclv reduces C2H2 to C2H4 plus C2H6, whereas AclMo only yields C2H4, a ...

Vanadium nitrogenase: A two-hit wonder?

“…Additionally, stopped-flow spectrometry shows that the rates of the association of the two component proteins and the MgATP-dependent electron transfer are very similar in the cases of the two nitrogenases, both too fast to be the rate-limiting step during the substrate turnover process. 34 There are, however, some differences between the catalytic behaviors of the two nitrogenases. At the standard temperature for both in vivo and in vitro assays ( i.e.…”

“…Additionally, stopped-flow spectrometry shows that the rates of the association of the two component proteins and the MgATP-dependent electron transfer are very similar in the cases of the two nitrogenases, both too fast to be the rate-limiting step during the substrate turnover process. 34 There are, however, some differences between the catalytic behaviors of the two nitrogenases. At the standard temperature for both in vivo and in vitro assays (i.e., 30 • C), the specific activity of Vnitrogenase is consistently lower than that of its Mo-counterpart, likely due to a reduced electron flux through the system; 5 yet, when the temperature is lowered (e.g., to 5 • C), V-nitrogenase becomes more active than Mo-nitrogenase.…”

Structure−Function Relationships of Alternative Nitrogenases

“…In the case of V-nitrogenase, only the reactions forming the first two steps of the catalytic cycle of nitrogenase turnover, i.e., the binding of MgATP to the Fe protein and subsequent electron transfer from Ac2 v to Ac1 v , have been studied. , The rate of electron transfer for the Ac2 v −Ac1 v complex ( k +2 = 46 ± 2 s -1 ) is significantly slower than for Ac2−Ac1 ( k +2 220 s -1 ). The dependence of this reaction on MgATP concentration ( K D = 230 ± 10 μM) and its competitive inhibition by MgADP ( K i MgADP = 30 ± 5 μM) were very similar to those of Mo-nitrogenase.…”