| The researches of nitrogenase have reached the atomic levels, but where the substrates bind and are reduced at the FeMoco remains unknown and the pathway of electron and proton transfer to FeMoco in the enzyme catalysis is also undetermined.In this research, site-directed mutagenesis and gene replacement procedures were used to construct two doubly altered MoFe proteins with a-Lysine 190 (a-Lys190) and a-Glutamine 194 (a-Gln194) substitutions and with a second substitution of citrate for homocitrate of FeMoco (from a nijV mutant) (a-Lys190/NifV- Kpl a-Gln194/NifV- Kp1) and two singly altered MoFe proteins with only a-Lysine 190 and a-Glutamine 194 substitutions (a-Lys190 Kpl and a-Gln194 Kpl) from Klebsiellapneumoniae. All four mutants expressing altered nitrogenases exhibited strictly Nif phenotype under N2-fixation condition and failed to grow diazotrophically. An auto-controlled fermentor was used to culture these strains under nitrogen fixation conditions and the nitrogneases were effectively depressed. Four altered MoFe proteins, together with WT and NifV" MoFe proteins and the WT Fe protein were purified by an identical procedure using chromatography and preparative electrophoresis. The activities of N2, H+ and C2H2 reduction catalyzed by these four MoFe proteins were detected and kinetics of H2 evolution and C2H2 inhibited by CO for some special altered MoFe proteins were also analyzed, which makes us possible inquire the N2 and H+ binding and reduced sites.Comparison of substrate-reduction properties of these altered MoFe proteins with WT and NifV-MoFe proteins showed that: (1) only the a-Gln194 substitution did not affect the total electron to proton reduction and notably double altered MoFe protein with substitution of citrate and a-Lys190 only retained very poor proton reduction activity. (2) a-Gln194 substitution made a more direct effect on N2 reduction then other two substitutions. (3) Substitution of a-Gln194 deflected electron to H2 evolution and substitution of a-Lys190 almost blocked the electron transfer to C2H2 reduction. (4) H2 evolution by MoFe protein with a-Gln194 and citrate substitutions was inhibited by CO at 32% inhibition rate, comparing to 71% of that for NifV- MoFe protein. (5) CO has a more weak affinity to MoFe protein with a-Lys190 substitutions then to NifV- MoFe protein or MoFe protein with a-Gln194 and citrate substitutions.Whole cell C2D2 reduction by all four mutants comparing to wild type and ni/V mutant was also detected. The result showed that only single a-Gln194 substitution did not perturb the stereospecificity of protonation of C2D2.The above comparing results indicate that in MoFe protein (1) a a-Gln190 site and its association with homocitrate are important for the transfer of electron/proton to FeMoco, while a-His194 site and the homocitrate are independent in H2 evolution. (2) a-Gln194 has a special role in the N2 reduction and acts as a regulatory site of electron distribution between H2 and NH3or between H2 and C2H4; (3) a-Glu190 and homocitrate share some common mechanism to affect a H2 evolution site of FeMoco.According the implications obtained in this study, the N2 and H+ binding and reduced model atFeMoco was proposed : (1) N2 binds and is reduced at the central Fe region of FeMoco, the electron and proton for N2 reduction is transferred from S2B atom of FeMoco via ct-Hism, the reaction is N2 + 8H+ +8e→ 2NH3 + H2, and is inhibited by CO. (2) Mo of FeMoco is occupied by an obligatory H2 evolution and the electron and proton to Mo is transferred from homocitrate, the reaction is 2H+ + 2e→ H2, and is escaped the inhibition by CO. a-Gln190 -omocitrate-Mo is an important electron transfer pathway to FeMoco.This N2 and H+ binding and reduced model also developed our early proposed two-site H2 evolution model for nitrogenase: Site I, N2-dependent H2 evolution site, is provided by the central six-Fe region of FeMoco, which is also provided the N2, C2H2, CO binding site. Site II, the N2-independent H2 evolution site, is located at the Mo...
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