No abstract

No abstract

No abstract

We have examined the reduction of cyanide by using the purified component proteins of nitrogenase (Av1 and Av2). The previously reported self-inhibition phenomenon was found to be an artifact. One of the two species present in cyanide solutions, CN-, was shown to be a potent reversible inhibitor (Ki = 27 microM) of total electron flow, apparently uncoupling MgATP hydrolysis and electron transfer. There appears to be no differential effect of CN- on the specific activities of Av1 and Av2 nor is there any apparent irreversible physical damage to Av2. CN- inhibition is completely reversed by low levels of CO, implying a common binding site. Azide partially relieves the inhibitory effect, but other substrates and inhibitors (N2, C2H2, N2O, H2) have no effect. The other species present in cyanide solutions, HCN, was shown to be the substrate (Km = 4.5 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough HCN concentration H2 evolution can be eliminated. The products are methane plus ammonia (six electrons), and methylamine (four electrons). There is an excess (relative to methane) of ammonia formed, which, according to electron balance studies, may arise from a two-electron intermediate. Both nitrous oxide and acetylene (but not N2) influence the distribution of cyanide reduction products, implying simultaneous binding. HCN appears to bind to and be reduced at an enzyme state more oxidized than the one responsible for either H2 evolution or N2 reduction.

The ability to fix dinitrogen is restricted to a small but diverse group of procaryotes that contain the nitrogenase system. This system is composed of two proteins called the Fe protein and the MoFe protein; the latter contains the site of N 2 binding and reduction. 1,2 The Fe protein contains a single Fe 4 S 4 cluster bridged between two identical subunits each of which has a single binding site for MgATP. It is the only known reductant capable of reducing the MoFe protein such that the latter can reduce substrates. Our current understanding of nitrogenase is based on in vitro experiments with purified nitrogenase proteins using dithionite as the electron donor. Under these conditions the Fe 4 S 4 cluster shuttles between the 2+ and 1+ oxidation states.In 1994, Watt and Reddy 3 reported that the [Fe 4 S 4 ] 1+ Fe protein could be reversibly reduced (E°′ ) -460 mV vs SHE) to the all-ferrous [Fe 4 S 4 ] 0 state. This reaction was reported to be pH independent between pH 7.0 and 8.0. [Fe 4 S 4 ] 0 could be produced using methylviologen, Ti(III)citrate, or the physiological electron donor flavodoxin as reductants but could not be generated when dithionite was used as a reductant. 1,3,4 The proposal for the formation of an [Fe 4 S 4 ] 0 state was based on two main lines of evidence. 3 First, Coulometric reduction of the [Fe 4 S 4 ] 1+ state of the Fe protein showed that a second electron could be added to the protein but did not show whether the reduction was metal-centered. 5 Second, the two-electron reduced Fe protein did not exhibit the S ) 1 / 2 EPR signal that arises from the [Fe 4 S 4 ] 1+ state, but that signal could be elicited by adding 1 equiv of oxidant to the fully reduced protein. These observations suggest, but do not prove, the unprecedented formation 6 of a protein-bound all-ferrous Fe 4 S 4 cluster.For the Mössbauer and EPR studies we produced the twoelectron reduced form of the Fe protein by treating 57 Fe-enriched Azotobacter Vinelandii Fe protein (AV2) with Ti(III) citrate. 8 Figure 1 shows two Mössbauer spectra of Ti(III) citrate-reduced AV2. The zero-field spectra exhibit quadrupole doublets down to 4.2 K. Preliminary analysis of the whole data set (30 spectra) revealed four distinct sites, all with isomeric shift δ ) 0.68 mm/s. The simulation of Figure 1A assumes four doublets of equal intensity with ∆E Q ) 1.25, 1.40, 1.75, and 3.08 mm/s. The absence of magnetic features in the 4.2 K zero-field spectrum strongly suggests that the Fe 4 S 4 cluster has integer or zero spin. The isomer shift, δ, is an excellent indicator for the oxidation state of an Fe 4 S 4 cluster; typically, the average value of δ increases by 0.10-0.12 mm/s per electron added. Table 1 shows that the isomeric shifts of all iron sites are larger than those of the ferrous pair (δ ) 0.59 mm/s 12 ) of the S ) 1 / 2 state of [Fe 4 S 4 ] 1+ AV2. From these observations we conclude that all iron sites of the cluster are ferrous.As shown in Figure 1B, a weak applied magnetic field elicits substantial 57 Fe magnetic hyperfine inter...

The basis of the chemiosmotic theory is that energy from light or respiration is used to generate a trans-membrane proton gradient. This is largely achieved by membrane-spanning enzymes known as 'proton pumps. There is intense interest in experiments which reveal, at the molecular level, how protons are drawn through proteins. Here we report the mechanism, at atomic resolution, for a single long-range electron-coupled proton transfer. In Azotobacter vinelandii ferredoxin I, reduction of a buried iron-sulphur cluster draws in a solvent proton, whereas re-oxidation is 'gated' by proton release to the solvent. Studies of this 'proton-transferring module' by fast-scan protein film voltammetry, high-resolution crystallography, site-directed mutagenesis and molecular dynamics, reveal that proton transfer is exquisitely sensitive to the position and pK of a single amino acid. The proton is delivered through the protein matrix by rapid penetrative excursions of the side-chain carboxylate of a surface residue (Asp 15), whose pK shifts in response to the electrostatic charge on the iron-sulphur cluster. Our analysis defines the structural, dynamic and energetic requirements for proton courier groups in redox-driven proton-pumping enzymes.

One of the most complex biosynthetic processes in metallobiochemistry is the assembly of nitrogenase, the key enzyme in biological nitrogen fixation. We describe here the crystal structure of an iron-molybdenum cofactor-deficient form of the nitrogenase MoFe protein, into which the cofactor is inserted in the final step of MoFe protein assembly. The MoFe protein folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits. In this structure, one of the three alpha subunit domains exhibits a substantially changed conformation, whereas the rest of the protein remains essentially unchanged. A predominantly positively charged funnel is revealed; this funnel is of sufficient size to accommodate insertion of the negatively charged cofactor.

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (⌬nifH MoFe protein) was purified in large quantity. The which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.The metalloenzyme nitrogenase complex catalyzes the biological reduction of dinitrogen to ammonia (for recent reviews, see Refs. 1-6). The enzyme is composed of two separately purifiable proteins, the iron (Fe) protein and the molybdenumiron (MoFe) 1 protein. The Fe Protein is a 60-kDa dimer of two identical subunits encoded by the nifH gene. The two subunits are bridged by a [4Fe-4S] cluster, and each subunit has a binding site for MgATP. The more complicated MoFe protein is a 230-kDa ␣ 2 ␤ 2 tetramer with the ␣ and ␤ subunits encoded by the nifD and nifK genes, respectively. The MoFe protein contains two different types of metal clusters, the [8Fe-7S] cluster (P-cluster) bridged between each ␣␤ subunit pair and the [Mo7Fe-9S-homocitrate] cluster (FeMoco) located within each ␣ subunits. Substrate reduction by the enzyme requires both component proteins, with the Fe protein serving as a specific reductant of the MoFe protein, which in turn provides the site of substrate reduction. To carry out the catalytic function of nitrogenase, the reduced Fe protein first binds two molecules of MgATP and undergoes a conformational change before forming a complex with the MoFe protein. Then, coupled with MgATP hydrolysis, electrons are transferred from the Fe protein to the P-clusters of the MoFe protein within the complex. This process is followed by the dissociation and re-reduction of the oxidized Fe protein and the dissociation of MgADP from the MoFe protein. Finally, the electrons are believed to be transferred from the P-cluster to the FeMoco, where substrate reduction occurs.FeMoco-deficient, but P-cluster containing MoFe proteins have proved to be useful for the study of two major aspects of the nitrogenase research, the maturation of MoFe protein (7-18) and the features of the P-cluster (