Protein nitrogenase

Iron-Sulfur Proteins Nitrogenase Metal Cluster Models. 

Iron Snlfur Proteins Nitrogenase Metal Clnster Models. 

The enzyme system for nitrogen fixation consists of two separate proteins. Nitrogenase (also called component I or molybdenum-iron protein) catalyzes the reduction of N2. The other, nitrogenase reductase (also called component II or iron protein), transfers electrons from ferredoxin or flavodoxin to nitrogenase (Figure 20.4)... 

The two components of the Fe-Mo protein nitrogenase, which is responsible for the fixation of nitrogen by microbes, have been purified and characterized from several sources. It seems that the enzyme acts through an elaborate mechanism which involves electron transport from one protein to the other promoted by reaction with Mg-ATP. Further results with a nitrogenase model have been reported. 

Many key protein ET processes have become accessible to theoretical analysis recently because of high-resolution x-ray stmctural data. These proteins include the bacterial photosynthetic reaction centre [18], nitrogenase (responsible for nitrogen fixation), and cytochrome c oxidase (the tenninal ET protein in mammals) [19, 20]. Although much is understood about ET in these molecular machines, considerable debate persists about details of the molecular transfonnations. 

The nitrogenase system reduces hundreds of millions of kilograms of nitrogen gas to ammonia each year, catalysing tire reaction at ambient temperatures and atmospheric pressure. Nitrogenase consists of two proteins tliat contain... 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

AH three nitrogenases comprise two separately puriftable component proteins. Each has a specific homodimeric Fe protein. The Fe... 

Fig. 4. Requirements, substrates, and products of Mo-nitrogenase catalysis, where I is the MoFe protein II the Fe protein and Pi is inorganic phosphate. The generating system is composed of creatine phosphate and creatine phosphokinase to recycle the inhibitory MgADP produced during catalysis to...

In contrast to the situation with the alternative nitrogenases, but with the notable exception of the C. pasteurianum proteins, the component proteins from aU. Mo-based nitrogenases interact as heterologous crosses to form catalyticaHy active enzymes (52). Carbon monoxide, CO, is a potent inhibitor of aU. nitrogenase-cataly2ed substrate reductions, with the exception of reduction (126). Molecular hydrogen has a unique involvement with Mo-nitrogenase... 

Fig. 6. View of the nitrogenase MoFe protein P-cluster pair where ( ) represents Fe, (O) S, and (Q) C as modeled (153). The side chain of one of the...

Fig. 7. View of the FeMo-cofactor prosthetic group of the nitrogenase MoFe protein with some of the surrounding amino acid residues where ( ) represents the molybdenum coordinated to a-His-442 and homocitrate (at the top), ( ) represents the iron, interspersed with the sulfur (O) and carbon...

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

These studies of protein-bound heterometallic cubanes have amply demonstrated that the heterometal site is redox active and able to bind small molecules. Although they have yet to be identified as intrinsic components of any protein or enzyme (except as part of the nitrogenase FeMo cofactor cluster (254)), they are clearly attractive candidates for the active sites of redox enzymes. 

Mr 220-250 kDa. Figure 1 shows an overall electron transfer pathway for the nitrogenases where the Fe proteins act as very specific, essential electron donors to the larger proteins. This is not the only role for the Fe proteins (see Section IV,C) and their role in the mechanism is almost certainly more complex than that of a simple electron transfer agent (see below. Section V). Electron transfer from the Fe protein to... 

In late 1992 the first crystal structures of the Fe and MoFe proteins of Mo nitrogenase frora Azotobacter vinelandii were published (1-3). 

The nitrogenase proteins are generally characterized by two letters indicating the species and strains of bacteria and the numerals 1 for the MoFe protein and 2 for the Fe protein. Thus, the Fe protein from Azotobacter vinelandii is Av2 and the MoFe protein from Klebsiella pneumoniae is Kpl. 

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