Mo-Fe protein

Extensive studies on nitrogenases, and in particular on the Fe-Mo protein (1) component of these complex enzymic systems, have revealed the presence of a unique Fe/Mo/S aggregate intimately involved in biological N2 fixation. 

Intense interest in the basic coordination chemistry of Fe/M/S clusters (M=Mo,W) has emerged in parallel with the advances in understanding the nature of the Fe/Mo/S center in the nitrogenases. Studies directed toward the synthesis of at least a structural analog for the Fe/Mo/S center have been guided by the broadly defined analytical and spectroscopic data on the Fe-Mo protein and the FeMoco. 

Spectroscopic studies on the Fe-Mo protein by EPR and Mossbauer spectroscopy have shown six iron atoms each in a distinctive magnetic environment coupled to an overall S=3/2 spin system (6,7,8) and electron nuclear double resonance (ENDOR) studies suggest one molybdenum per spin system (8). The 5 Fe signals (five or six doublets) observed in the ENDOR spectra (8) indicate a rather asymmetric structure for the Fe/Mo/S aggregate in which the iron atoms roughly can be grouped into two sets of trios, each set having very similar hyperfme parameters. 

Extended X-ray absorption fine structure (EXAFS) studies on the Fe/Mo/S aggregate in nitrogenase have made available structural data that are essential in the design of synthetic analog clusters. Analyses of the Mo K-edge EXAFS of both the Fe-Mo protein and the FeMoco (9) have shown as major features 3-4 sulfur atoms in the first coordination sphere at 2.35 A and 2-3 iron atoms further out from the Mo atom at 2.7 A. The Fe EXAFS of the FeMoco (10,11) shows the average iron environment to consist of 3.4 1.6 S(C1) atoms at 2.25(2) A, 2.3 +0.9 Fe atoms at 2.66(3) A, 0.4 0.1 Mo atoms at 2.76(3) A and 1.2 1.0 0(N) atoms at 1.81(7) A. In the most recent Fe EXAFS study of the FeMoco (11) a second shell of Fe atoms was observed at a distance of 3.75 A. 

The N2-fixing enzyme used by the bacteria is nitrogenase. It consists of two components an Fe protein that contains an [Fe4S4] cluster as a redox system (see p. 106), accepts electrons from ferredoxin, and donates them to the second component, the Fe-Mo protein. This molybdenum-containing protein transfers the electrons to N2 and thus, via various intermediate steps, produces ammonia (NH3). Some of the reducing equivalents are transferred in a side-reaction to In addition to NH3, hydrogen is therefore always produced as well. 

The eight-step nitrogenase cycle. Nitrogenase contains two complex protein components component I and component II. The nitrogenase cycle starts when component II (an Fe—protein) binds to component I (usually an Fe—Mo—protein). The binding requires two ATP molecules. During the bound state component II transfers an electron to component I. This is followed by the hydrolysis of the two ATPs and the separation of the two components that begin a new cycle. In... 

All species that can fix nitrogen possess the nitrogenase complex. Its structure, similar in all species so far investigated, consists of two proteins called dinitro-genase and dinitrogenase reductase. Dinitrogenase (240 kD), also referred to as Fe-Mo protein, is an a -heterotetramer that contains two molybdenum (Mo) atoms, and 30 iron atoms. It catalyzes the reaction N2 + 8 H+ + 8 —> 2... 

A cartoon representation of nitrogenase in operation, showing the component proteins. The active site of nitrogenase that resides in the Fe—Mo protein determined from a crystal structure analysis, and which contains two linked distorted M4S4 cubes, is also shown. 

Two binding sites for acetylene have been detected by e.p.r. studies on the (Fe-Mo) protein of nitrogenase from Klebsiella pneumoniae. The reduction product, ethylene, has a single site and only the stronger acetylene site with an association constant in excess of 6 x 10 M at pH 7.4 and 10 °C is catalytically active. Binding of acetylene or carbon monoxide at the second site results in a dead-end complex. 

Thiol extrusion of the FeS centres from Azobacter vinelandii and Clostridium pasteurianum with alkyl fluorothiols allows identification of at least two Fe4S4 and one FegSj cluster in the (Fe-Mo) protein. A cluster model in which two Fe4S4 units are bridged through a comer iron on each cube by a Mo S4 unit is proposed for the active site of the (Fe-Mo) cofactor. Molecular nitrogen binds axially to the central molybdenum and is Ji-bonded to the iron in the cubes. This weakens and activates the N=N bond and electrons are injected stepwise via the two iron-sulphur cubes with successive protonation at the terminal N. This latter point is consistent with a report that N—NHg is important in nitrogen fixation. 

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. 

It relies on a complicate biological electron route for electron to transfer from electron-donator to N2 (adsorption). The transfer orientation is electron-donator —> Fe redox protein Fe protein —> Fe-Mo protein. It requires ATP-Mg + to activate electron in order to ensure the smoothness of the electrical route. ATP itself hydrolyzes to ADP during this process. H+ may be transferred to active center by side-groups (—SH, imidazole, —OH) on proteins. 

A relationship between the redox state of an iron—sulfur center and the conformation of the host protein was furthermore established in an X-ray crystal study on center P in Azotobacter vinelandii nitroge-nase (270). In this enzyme, the two-electron oxidation of center P was found to be accompanied by a significant displacement of about 1 A of two iron atoms. In both cases, this displacement was associated with an additional ligation provided by a serine residue and the amide nitrogen of a cysteine residue, respectively. Since these two residues are protonable, it has been suggested that this structural change might help to synchronize the transfer of electrons and protons to the Fe-Mo cofactor of the enzyme (270). 

Reichenbecher W, A Rudiger, PMH Kroneck, B Schink (1996) One molecule of molybdopterin guanine dinucleotide is associated with each subunit of the heterodimeric Mo-Fe-S protein transhydroxylase of Pelobacter acidigalUci as determined by SDS (PAGE) and mass spectrometry. Eur J Biochem 237 406-413. 

Concurrently with the X-ray crystallographic studies, extended X-ray absorption fine structure (EXAFS) studies confirmed many of the bond distances proposed for nitrogenase s FeMoco cluster. The EXAFS data of reference 25 indicate short Fe-Fe distances of 2.61, 2.58, and 2.54 A for M+, M (resting state), and M forms, respectively. The authors believe that the short M center bond lengths indicate Fe-Fe bonds in this cluster. In another study using dithionite-reduced MoFe-protein Fe-S, Fe-Fe, Fe-Mo distances of 2.32, 2.64, and 2.73 A, respectively, were found in the 1 to 3 A region and Fe-Fe, Fe-S and Fe-Fe distances of 3.8, 4.3, and 4.7 A, respectively, were found in the 3 to 5 A region.30... 

Nitrogenases from various nitrogen fixing organisms seem to contain the same Fe/Mo/S structural unit that occurs as an extractable cofactor (FeMoco) (2). Extracts of the Fe-Mo component protein from inactive mutant strains of different microorganisms that do not contain the Fe/Mo/S center are activated upon addition of the FeMoco. 

Xanthine oxidase, which requires Fe, Mo and flavin adenine dinucleotide (FAD) as co-factors, is capable of oxidizing lipids via the production of superoxide radicals. It represents about 20% of the MFGM protein and part is readily lost from the membrane, e.g. on cooling isoelectric focusing... 

Hydration and/or dehydration reactions are frequently catalyzed by metallopro-teins. Examples are proteins containing nickel (urease), zinc (e.g., peptidases), molybdenum (the hydratase partial reaction of formate oxidoreductase), tungsten (acetylene hydratase). An obvious difference between Ni, Zn, on the one hand, and Fe, Mo, W, on the other, is that the first are directly coordinated to the protein whereas the latter are also part of a cofactor. With reference to the Fe/S cluster in aconitase it has been suggested that cofactor coordination may provide an added flexibility to the active site, in particular to the substrate binding domain [15],... 

The conversion of dinitrogen to ammonia is one of the important processes of chemistry. Whereas the technical ammonia synthesis requires high temperature and pressure (1), this reaction proceeds at room temperature and ambient pressure in nature, mediated by the enzyme nitrogenase (2). There is evidence that N2 is bound and reduced at the iron-molybdenum cofactor (FeMoco), a unique Fe/Mo/S cluster present in the MoFe protein of nitrogenase. Although detailed structural information on nitrogenase has been available for some time (3), the mechanism of N2 reduction by this enzyme is still unclear at the molecular level. Nevertheless, it is possible to bind and reduce dinitrogen at simple mono- and binuclear transition-metal systems which allow to obtain mechanistic information on elemental steps involved... 

C. Proposed Structures. That the FeMo-cofactor is a structurally novel cluster containing Fe, Mo, and S is suggested by its composition and spectroscopic properties, by the observation that it is resistant to conditions that destroy (i.e., dilute acid50,67 or 80% (v/v) 5M imidazole in hexamethylphosphoramide14 ) or react with66 normal protein-... 

The [2Fe 2S], [3Fe S], and [4Fe S] clusters that are found in simple Fe S proteins are also constituents of respiratory and photosynthetic electron transport chains. Multicluster Fe S enzymes such as hydrogenase, formate dehydrogenase, NADH dehydrogenase, and succinate dehydrogenase feed electrons into respiratory chains, while others such as nitrate reductase, fhmarate reductase, DMSO reductase, and HDR catalyze the terminal step in anaerobic electron transport chains that utihze nitrate, fumarate, DMSO, and the CoB S S CoM heterodisulfide as the respiratory oxidant. All comprise membrane anchor polypeptide(s) and soluble subunits on the membrane surface that mediate electron transfer to or from Mo cofactor (Moco), NiFe, Fe-S cluster or flavin active sites. Multiple Fe-S clusters define electron transport pathways between the active site and the electron donor or... 

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