Isolated FeMoCo Catalytic Reactivity In Nonprotein Surrounding Substrate And Inhibitor Interactions C2H2 N2 CO

T.A. Bazhenova, M.A. Bazhenova, G.N. Petrova

Inst, of Problems of Chem. Phys., Russ. Acad. Sci., 142432, Chernogolovka, Russia

To understand the mechanism of substrate reduction in the active site of nitrogenase the catalytic reactivity of the isolated FeMo-co with respect to C2H2, and/or CO, and/or N2 reduction in non-enzymatic surroundings (DMF, Eu/Hg, PhSH) was investigated. The quality of the FeMo-co extracted (before and after catalytic reactions) was evaluated from its Fe:Mo ratio and its ability to reconstitute the activity of FeMo-co-deficient MoFe protein (NifB-Kpl) in crude extracts of Klebsiella pneumoniae Kp5058. It has been found that the structural integrity of FeMo-co was retained during these reactions. Study of a steady-state kinetics of C2H2 reduction catalyzed by FeMo-co extracted has been carried out. It has been found in particular that the FeMo-co cluster reduced by Eu/Hg demonstrates substrate induced cooperativity among two sites capable to bind and reduce acetylene (Bazhenova et al. 2000). The bell-shaped profile was observed for dependence of C2H2 reduction on PhSH concentration. The C2D2 reduction stereospecificity was examined by FTIR spectroscopy. Q5-C2D2H2 was shown to be the main product (Bazhenova et al. 2001).

Carbon monoxide (CO) is not reduced in this system, but it is a potent inhibitor of C2H2 reduction catalyzed by isolated FeMo-co. For CO the type of inhibition was shown to be reversible and competitive rather than noncompetitive (Bazhenova et al. 2001). The inhibition constants for ethylene and ethane formation were found to be different: K (atm) = 0.004 for C2H4 and 0.009 for C2H6. Distinct C2H2 bonding sites are inhibited by CO differently.

We have found that at low, unsaturating, C2H2 pressure, dinitrogen inhibits acetylene reduction catalyzed by FeMo-co in DMF solution with Eu/Hg as a reducing agent. The type of inhibition was shown to be competitive and reversible, Kt = 0.49 atm N2 both for C2H4 and C2H6 formation (Bazhenova 2001). The value of N2 inhibition constant for acetylene reduction catalyzed by the isolated FeMo-co is similar to those for wild-type, al95Gln (Kim C-H et al. 1995) and al95Asn (Fisher et al. 2000) nitrogenases. We therefore conclude that it is possible to obtain in non-enzymatic conditions a state of the isolated cofactor which is capable of binding the N2 molecule.

On the basis of these results we concluded that the isolated FeMo-co being reduced by Eu/Hg has two distinct substrate and inhibitor binding sites. One of these (site 1) has a high affinity for C2H2 binding and reduction (Km = 0.006 atm C2H2) and can also bind reversibly N2 or CO molecules without reduction. The other site (site 2) has a much lower affinity for C2H2 binding (the effective Km for both two C2H2 binding sites is 0.08 atm) and can also bind CO but not N2.

The analysis of all results obtained shows that the isolation of FeMo-co out of the protein matrix results in a loss of the ability to catalyze the reduction of Nf, the capability to reduce acetylene and protons, and the capability to coordinate reversibly N2 molecule under the action of appropriate chemical reducing agent is conserved practically unchanged.

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