Electron Transfer Pathways In The Process Of N₂and H⁺Reduction Catalyzed By Nitrogenases

Biological nitrogen fixation, Ihe reduction of dinitrogen (N2) to two ammonia molecules (NH3), is mainly catalyzed by the Mo-dependent nitrogenase. This process utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. It is reported that more than1mol H2was released as1mol N2was reduced, based on the kinetic study of nitrogenases from three different microbes. In this process, there should be more than two different ET pathways between P-cluster and FeMoco for the energy supply. Then, an ET pathway from P-cluster to the S2B atom of FeMoco was pointed out in our lab previously. In this study, the other ET pathways were explored by the combination of theoretical calculation and biological methods.Based on the survey of the surroundings between P-cluster and FeMoco in MoFe protein from Klebsiella oxytoca (PDB ID:1QGU), a proposed ET pathway was found by SPDBV4.01program, which flows Ihrough P-clusterâ†'β93Cysâ†'β-1022Asnâ†'β-107Gluâ†'α-431Lysâ†'α-423Ileâ†'homocitrate (O4atom)â†'FeMoco (Mo atom). And β-102Asn, α-431Lys and α-423Ile were considered to be the key points. Then, in order to map this ET pathway, three site-directed mutants were constructed in K. oxytoca M5al strain (termed Nβ102A, Ka431H and Ia423P), in which these three residues of the MoFe protein were replaced by alanine, histidine and proline, respectively. In the flasks culture, Ia423P was the only strain which cannot grow under nitrogen-fixation condition and the specific activity of C2H2reduction was pretty low, while the growth and the maximal specific activities of the other two mutants were rough the same as these of wild-type strain. Furthermore, Ihe relative expression level of the nifD gene in Ia423P strain was4-fold of that in wild-type. Thus, the characteristics of Ia423P strain shown above were not due to blocking of gene transcription resulting from residue substitution, but may have been due to the decreased catalytic ability of the enzyme itself. Both MoFe and Fe proteins were purified from Iα423P variant and wild type strain by sequential anion exchange chromatography and preparative electrophoresis respectively. The maximal C2H2-reduction and H+-reduction activities of Ia423P MoFe protein were13%and21%as those of wild-type MoFe protein respectively, indicating that the substitution at α-423Ile reduced the catalytic ability of Iα423P MoFe protein. What is more, the EPR (Electron Paramagnetic Resonance) spectra of Iα423P-MoFe protein in resting state showed the content of FeMoco in this variant protein was lower, which illustrated that α-423Ile plays a fundamental role in structure assembling of MoFe protein. It was also found that, both the substitution in Ia423P strain ((α-423Ileâ†'Pro) and the change in UNF837strain (homocitrateâ†'citrate) would destroy or decrease the interaction between a-423and homocitrate theoretically, but the specific activities of double-site mutants were not exactly the same when α-194His was replaced by glutamine in Ia423P and UNF837 strain. In addition, when both α-423Ileand homocitrate were changed, the double-site mutant UN423P exhibited even lower ability in reducing C2H2than wild-type strain did. It is proposed that α-423Ile serves as a gate to the Mo site of FeMoco.By focusing on the whole crystal structure of MoFe protein from Azotobacter vinelandii (PDB ID:3U7Q), a new method was employed to calculate the possible ET pathways during nitrognen-fixation, and three different pathways were pointed out. In addition, it was more complicated to compare the ET-pathway change with different substitutions at α-191Gln and α-65Ala separately. In order to exam the role of α-191Gln, seven alternated-MoFe proteins from mutants constructed by replacing with different residues were gained and characterized. It was shown that the specific activities of all variant-MoFe proteins were decreased, while the primary electron transfer is slow down from the stopped-flow traces change at430nm. The result indicated that α-191Gln is essential for transferring electrons to FeMoco.In summary, it is proved that both α-423Ileand α-191Glnare the bridges in delivering electrons from P-cluster to FeMoco. For details, α-191Gln is located at the ET pathway from P-cluster to the S2B atom of FeMoco, supplying for N2reduction coupled with H2formation, whereas α-423Ile responds as an entry via homocitrate to the Mo atom of FeMoco only for H+reduction. This study supplied the experimental data for the ’dual reduction-site’ model, and provided critical insights into the further study of the catalytic reaction mechanism of nitrogenase.