Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp.

Ammonium chloride (greater than or equal to 0.05 mM) effectively and reversibly inhibited the nitrogenase activity of Azospirillum brasilense, Azospirillum lipoferum and Azospirillum amazonense. The glutamine synthetase inhibitor L-methionine-DL- sulfoximine abolished this "switch-off" in A. lipoferum and A. brasilense, but not in A. amazonense. Azaserine, an inhibitor of glutamate synthase, inhibited nitrogenase activity itself. This provides further evidence for glutamine as a metabolite of regulatory importance in the NH4+ switch-off phenomenon. In A. brasilense and A. lipoferum, a transition period before the complete inhibition of nitrogenase activity after the addition of 1 mM ammonium chloride was observed. The in vitro nitrogenase activity also was decreased after treatment with ammonium. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a second dinitrogenase reductase (Fe protein) subunit appeared, which migrated in coincidence with the modified subunit of the inactive Fe protein of the nitrogenase of Rhodospirillum rubrum. After the addition of ammonium 32P was incorporated into this subunit of the Fe protein of A. brasilense. In A. amazonense, the inhibition of nitrogenase activity by ammonium was only partial, and no transition period could be observed. The in vitro nitrogenase activity of ammonium-treated cells was not decreased, and no evidence for a modified Fe protein subunit was found. Nitrogenase extracts of A. amazonense were active and had an Fe protein that migrated as a close double band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins.

A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.     

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii.

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.     

The vanadium-containing nitrogenase of Azotobacter

Fifty years after a role of vanadium in biological fixation was proposed, it was shown that in addition to their well-characterized molybdendum nitrogenases, Azotobacter chroococcum and Azotobacter vinelandii both have a genetically distinct nitrogenase system in which the conventional molybdoprotein is replaced by a vanadoprotein. Both Mo-nitrogenases and V-nitrogenases have similar requirements for activity: MgATP, a low potential reductant and the absence of oxygen. The genes encoding the V-nitrogenase are expressed only under conditions of Mo-deficiency. V-Nitrogenase of A.chroococcum is made up of a tetrameric VFe protein (Mr 210,000) with an alpha 2 beta 2 structure containing two V atoms, 23 Fe atoms and 20 acid-labile sulphide atoms per tetramer, and a dimeric Fe protein (Mr 64,000) with a gamma 2 structure containing four Fe atoms and four acid-labile sulphide atoms per dimer. Vanadium K-edge X-ray absorption spectroscopy indicates that V in the VFe protein, like Mo in MoFe protein, has S, Fe and possibly O as nearest neighbours. A vanadium- and iron-containing cofactor (FeVaco) can be extracted from the VFe protein and will restore C2H2 reductase, but no nitrogenase activity, to the inactive MoFe protein accumulated by mutants unable to synthesize the molybdenum- and iron-containing co-factor of Mo-nitrogenase. The products of C2H2 reduction by the hybrid protein (C2H6 as well as C2H4) are a characteristic of the VFe protein and provide evidence that FeVaco is, or forms part of the active site of V-nitrogenase.     

Properties of the nitrogenase system from a photosynthetic bacterium, Rhodospirillum rubrum.

Soluble nitrogenase from Rhodospirillum rubrum has been isolated and separated into its two components, the MoFe protein and the Fe protein. The MoFe protein has been purified to near homogeneity and has a molecular weight or 215 000. It contains two Mo, 25--30 Fe and 19--22 acid-labile sulphide and consists of four subunits, Mw 56 000. The Fe protein has a molecular weight 65 000. It contains approximately four Fe and four acid-labile sulphide and consists of two subunits, Mw 31 500. The highest specific activities for the purified components are 920 and 1260 nmol ethylene produced per min per mg protein, respectively. The purified components require the membrane component for activity (Nordlund, S., Eriksson, U. and Baltscheffsky, H. (1977) Biochim. Biophys. Acta 462, 187--195). Titration of the MoFe protein with the Fe protein shows saturation and excess MoFe protein over Fe protein is inhibitory. Addition of Fe2+ or Mn2+ to the reaction mixture increases the activity apparently through interaction with the membrane component.     

Purification and Characteristics of Mn-containing Nitrogenase Component

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but undergo phenotypic reversal to Nif+ under Mo deficiency, was able to grow in Mo- and NH3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C2H2- and H+-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰprotein.     

Pectin decomposition and associated nitrogen fixation by mixed cultures of Azospirillum and Bacillus species.

Cocultures of different Azospirillum species with Bacillus polymyxa or Bacillus subtilis allow the efficient utilization of pectin as carbon and energy sources for nitrogen fixation. The nitrogenase activity obtained with cocultures was as high as 30-80 nmol C2H4 h-1 mL-1, a much higher value than that obtained with pure cultures of either Azospirillum (up to 13 nmol C2H4 h-1 mL-1) or B. polymyxa (up to 2 nmol C2H4 h-1 mL-1) alone. To establish to what extent each partner contributed to nitrogenase activity, acetylene reduction was assayed as a function of time and it was also measured on Azospirillum cultivated in the cultures filtrates of the Bacillus. The results suggested that the nitrogenase activity was mostly produced by Azospirillum. The nitrogenase activity occurred at the expense of the degradation and fermentation products of the pectin. The new pectinolytic species, Azospirillum irakense, utilized both degradation and fermentation products of pectin, whereas the nonpectinolytic strains (Azospirillum brasilense, Azospirillum lipoferum, Azospirillum amazonense) utilized only the fermentation products of pectin, including acetic and succinic acids. These cocultures can be considered as metabolic associations, where the Bacillus produces degradation and fermentation products of pectin, which can be used by Azospirillum species.     

Electron allocation to alternative substrates of Azotobacter nitrogenase is controlled by the electron flux through dinitrogenase

The electron flux through dinitrogenase (MoFe protein, protein containing Mo and Fe) from Azotobacter vinelandii controls the relative effectiveness of alternative substrates as electron acceptors in the nitrogenase system. The electron flux through dinitrogenase reductase (Fe protein) or the concentration of MgATP do not directly control electron allocation but rather control it via their influence on the electron flux through dinitrogenase. Kinetic properties of substrate reduction were studied as a function of the electron flux through dinitrogenase. N₂ was most effective at high electron fluxes, whereas H⁺ was the most effective acceptor at very low rates of electron flow through dinitrogenase. The K_m for acetylene was dependent on the electron flux through dinitrogenase, whereas the K_m for N₂ was much less sensitive to this electron flux. The lag period before the onset of acetylene reduction was proportional to the turnover time of dinitrogenase, and was approx. 12 times greater than the dinitrogenase turnover time. pH has effects on the electron allocation to substrates beyond that expected from the effect of pH on the electron flux; thus, pH may alter the relative ability of the nitrogenase enzyme system to reduce alternative substrates.     

Purification and characterization of the alternative nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum.

The alternative nitrogenase from a nifH mutant of the photosynthetic bacterium Rhodospirillum rubrum has been purified and characterized. The dinitrogenase protein (ANF1) contains three subunits in an apparent alpha2beta2gamma2 structure and contains Fe but no Mo or V. A factor capable of activating apo-dinitrogenase (lacking the FeMo cofactor) from Azotobacter vinelandii was extracted from the alternative dinitrogenase protein with N-methylformamide. The electron paramagnetic resonance (EPR) signal of the dinitrogenase protein is not characteristic of the EPR signals of molybdenum- or vanadium-containing dinitrogenases. The alternative dinitrogenase reductase (ANF2) was purified as an alpha2 dimer containing an Fe4S4 cluster and exhibited an EPR spectrum characteristic of dinitrogenase reductases. The enzyme complex reduces protons to H2 very well but reduces N2 to ammonium poorly. Acetylene is reduced to a mixture of ethylene and ethane.     

Hydrolysis of nucleoside triphosphates other than ATP by nitrogenase

The hydrolysis of ATP to ADP and P(i) is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron (Fe) protein and the molybdenum-iron (MoFe) protein. The oxidation state of nitrogenase was found to greatly influence the nucleotide hydrolysis rates. MgATP hydrolysis rates were 20 times higher under dithionite reducing conditions (approximately 4,000 nmol of MgADP formed per min/mg of Fe protein) as compared with indigo disulfonate oxidizing conditions (200 nmol of MgADP formed per min/mg of Fe protein). In contrast, MgGTP, MgITP, and MgUTP hydrolysis rates were significantly higher under oxidizing conditions (1,400-2,000 nmol of MgNDP formed per min/mg of Fe protein) as compared with reducing conditions (80-230 nmol of MgNDP formed per min/mg of Fe protein). The K(m) values for MgATP, MgGTP, MgUTP, and MgITP hydrolysis were found to be similar (330-540 microM) for both the reduced and oxidized states of nitrogenase. Incubation of Fe and MoFe proteins with each of the MgNTP molecules and AlF(4)(-) resulted in the formation of non-dissociating protein-protein complexes, presumably with trapped AlF(4)(-) x MgNDP. The implications of these results in understanding how nucleotide hydrolysis is coupled to substrate reduction in nitrogenase are discussed.     

Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum.

In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe--2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO--Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of 'conformational protection' in this organism.     

Characteristics and Crystallization of Nitrogenase MoFe Protein from a nif Z Deleted Strain of Azotobacter vinelandii

△nifZ MoFe protein purified from a nifZ deleted strain of Azotobacter vinelandii (DJ194) was shown to be pure by SDS-Polyacrylamide gel electrophoresis. The protein contained 1.5 Mo atoms and 15.9 Fe atoms per molecule, the ratio of Fe to Mo was lower than that of the MoFe protein purified from the wild type strain of A. vinelandii; and Call2, H+ -reduction activity and their ratio (C2H4/H2 (Ar)) were 16.6%, 21.7% and 77.2% of those of the wild type MoFe protein, respectively. Under a somewhat different condition from that for the crystallization of the wild type MoFe protein dark brown rhombohedron crystals of △nifZ MoFe protein were obtained. It indicated that the deletion of the △nif Z resulted in the decrease of number or change in the structure of P-cluster in the mutant MoFe protein, which caused the significant structured and function of change of the protein.     

Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense.

The Azospirillum brasilense draT gene, encoding dinitrogenase reductase ATP-ribosyltransferase, and draG gene, encoding dinitrogenase reductase activating glycohydrolase, were cloned and sequenced. Two genes were contiguous on the A. brasilense chromosome and showed extensive similarity to the same genes from Rhodospirillum rubrum. Analysis of mutations introduced into the dra region on the A. brasilense chromosome showed that mutants affected in draT were incapable of regulating nitrogenase activity in response to ammonium. In contrast, a mutant with an insertion in draG was still capable of ADP-ribosylating dinitrogenase reductase in response to ammonium but was no longer able to recover activity after ammonium depletion. Plasmid-borne draTG genes from A. brasilense were introduced into dra mutants of R. rubrum and restored these mutants to an apparently wild-type phenotype. It is particularly interesting that dra mutants of R. rubrum containing draTG of A. brasilense can respond to darkness and light, since A. brasilense is a nonphotosynthetic bacterium and its dra system does not normally possess that regulatory response. The nifH gene of A. brasilense, encoding dinitrogenase reductase (the substrate of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase), is located 1.9 kb from the start of draT and is divergently transcribed. Two insertion mutations in the region between draT and nifH showed no significant effect on nitrogenase activity or its regulation.     

Posttranslational regulatory system for nitrogenase activity in Azospirillum spp.

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.     

Molybdenum and vanadium nitrogenases of Azotobacter chroococcum. Low temperature favours N2 reduction by vanadium nitrogenase.

A comparison of the effect of temperature on the reduction of N2 by purified molybdenum nitrogenase and vanadium nitrogenase of Azotobacter chroococcum showed differences in behaviour. As the assay temperature was lowered from 30 degrees C to 5 degrees C N2 remained an effective substrate for V nitrogenase, but not Mo nitrogenase, since the specific activity for N2 reduction by Mo nitrogenase decreased 10-fold more than that of V nitrogenase. Activity cross-reactions between nitrogenase components showed the enhanced low-temperature activity to be associated with the Fe protein of V nitrogenase. The lower activity of homologous Mo nitrogenase components, although dependent on the ratio of MoFe protein to Fe protein, did not equal that of V nitrogenase even under conditions of high electron flux obtained at a 12-fold molar excess of Fe protein.     

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.     

固氮菌接种对棉苗氮磷钾吸收、某些酶活性及产量的影响(英文)

Spermospheremodels和盆栽试验结果表明 ,海岛棉 (GossypiumbarbadenseL .)苗接种自生固氮菌(Azotobactersp .)、巴西固氮螺菌NO40 (AzospirillumbrasilenseNO40 )、多粘芽孢杆菌 (BacilluspolymyxaCF)和根瘤菌 (Rhizobium) ,和以自生固氮菌分别与其它 3种供试菌种两者的混合菌 ,能增强棉花根际固氮酶活性和棉苗对氮的吸收 ,提高功能叶中氮、磷和叶绿素含量 ,从而有利于提高生物学产量 ,尤以自生固氮菌的促进效应最为显著。另一方面 ,混合菌处理较单一菌株处理 ,可以显著提高棉苗对氮的吸收 ,增加干物质积累提高皮棉产量 ,其中尤以固氮菌分别与根瘤菌或巴西固氮螺菌NO40的协同效应最显著     

Distinct structural features of the alpha and beta subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences

Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein. Structural genes (nifD and nifK) encoding alpha and beta subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced. The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein. Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD. An open reading frame following nifK was identified as nifE. The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated beta subunit. Cp nifK encodes a polypeptide of 458 amino acid residues (Mr 50 115) whose amino-terminal region is about 50 residues shorter than the otherwise conserved corresponding polypeptides from four other organisms. In contrast, Cp alpha subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein. It therefore appears that the combined size of the alpha and beta subunits could be important to nitrogenase function. An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C. pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine residues, differentiating the Cp MoFe protein from others. These different regions may be further tested for correlation with distinct properties of Cp nitrogenase.     

Purification and Characterization of Cr-containing Nitrogenase Component I

A mutant UW3, which is unable to fix N2 in the presence of Mo (Nif-) but can undergo phenotypic reversal to Nif+ under Mo deficient conditions, was able to grow in Cr containing but Mo and NH3 deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW3 grown on the Cr containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C2H2 and H+ reduction activity of MoFe protein from the wild type strain of Azotobacter vinelandii Lipmann. The Cr containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two differentsubunits (α2β2). The preliminary results indicated that the Cr containing protein might be a nitrogenase component Ⅰ protein.     

Iron-sulfur clusters in the molybdenum-iron protein component of nitrogenase. Electron paramagnetic resonance of the carbon monoxide inhibited state.

Carbon monoxide inhibits reduction of dinitrogen (N2) by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 X 10(-4) and 4.5 X 10(-4) and 7 X 10(-4) atm and 14 X 10(-4) atm for the two enzymes, respectively). The onset of inhibition is within the turnover time of the enzyme, and CO does not affect the electron flux to the H2-evolving site. The kinetics of CO inhibition of N2 reduction are simple, but CO inhibition of acetylene reduction is complicated by substrate inhibition effects. When low-temperature (approximately 13 K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, it is found that low concentrations of CO ([CO] = [enzyme]) induce the appearance of a signal with g values near 2.1, 1.98, and 1.92 with t1/2 approximately 4 s, while higher concentrations of CO lead to the appearance of a signal with g values near 2.17, 2.1, and 2.05 with a similar time course. The MoFe proteins from Rhizobium japonicum and Rhodospirillum rubrum, reduced with Azotobacter Fe protein in the presence of CO, give similar results. Under conditions which promote the accumulation of H2 in the absence of CO, an additional EPR signal with g values near 2.1, 2.0, and 1.98 is observed. The use of Azotobacter nitogenase components enriched selectively with 57Fe or 95Mo, as well as the use of 13CO, permitted the assignment of the center(s) responsible for the induced signals. Only 57Fe, when present in the MoFe protein, yielded broadened EPR signals. It is suggested that the MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferrodoxins. It is further proposed that the CO-induced signals arise from states of the MoFe protein in which CO inhibits electron flow to the N2-reducing site so that the iron-sulfur cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic states of high-potential iron-sulfur proteins and ferredoxins, respectively. The "no-CO" signal may be either an additional center or the N2-reducing site with H2 bound competitively.