Localization of a substrate binding site on the FeMo-cofactor in nitrogenase: trapping propargyl alcohol with an alpha-70-substituted MoFe protein

Substitution of the MoFe protein alpha-70(Val) residue with Ala or Gly expands the substrate range of nitrogenase, allowing the reduction of larger alkynes, including propargyl alcohol (HC[triple bond]CCH(2)OH). Herein, we report characterization of the alpha-70(Val)(-->)(Ala) MoFe protein with propargyl alcohol trapped at the active site. The alpha-70(Ala) variant MoFe protein was rapidly frozen during reduction of propargyl alcohol, resulting in the conversion of the resting-state FeMo-cofactor EPR signal (S = 3/2 and g = [4.41, 3.60, 2.00]) to a new state (S = 1/2 and g = [2.123, 1.998, 1.986]). This EPR signal of the new state increased in intensity with increasing propargyl alcohol concentration, consistent with the binding of a single substrate. The EPR signal of the propargyl alcohol state showed temperature and microwave power dependencies markedly different from those of the classic FeMo-cofactor EPR signal, consistent with the difference in spin. The new state is analogous to that induced by the binding of the inhibitor CO ("lo CO" state) to FeMo-cofactor in the wild-type MoFe protein. The (13)C ENDOR spectrum of the alpha-70(Ala) MoFe protein with trapped (13)C-labeled propargyl alcohol exhibited three well-resolved (13)C doublets centered at the (13)C Larmor frequency with isotropic hyperfine couplings of approximately 3.2, approximately 1.4, and approximately 0.7 MHz, indicating that the alcohol (or a fragment) is coordinated to the cofactor. The results presented here localize the binding site of propargyl alcohol to one [4Fe-4S] face of FeMo-cofactor and indicate roles for the alpha-70(Val) residue in controlling FeMo-cofactor reactivity.     

Diazene (HN=NH) is a substrate for nitrogenase: insights into the pathway of N2 reduction

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N2 reduction, is inhibited by H2. This contrasts with the absence of H2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with alpha-195His substituted by Gln and alpha-70Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3/2 resting state FeMo-cofactor to a novel S = 1/2 state with g1 = 2.09, g2 = 2.01, and g3 approximately 1.98. 15N- and 1H-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N2 by nitrogenase. Implications of these findings for the mechanism of N2 reduction by nitrogenase are discussed.     

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC triple bond CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein alpha-70(Val) residue, whose side chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of alpha-70(Val) by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC triple bond C-CH(3)), whereas, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the alpha-70(Val) residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC triple bond C-CH(2)-CH(3)). This protein shows no detectable reduction of the internal alkyne 2-butyne (H(3)C-C triple bond C-CH(3)). In contrast, substitution of the nearby alpha-191(Gln) residue by alanine, in combination with the alpha-70(Ala) substitution, does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.     

Substrate interaction at an iron-sulfur face of the FeMo-cofactor during nitrogenase catalysis

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N(2) to 2NH(3). Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC triple bond C-CH(2)OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid alpha-70(Val). Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC triple bond CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H(2)N-NH(2)) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (alpha-70(Val)) MoFe protein. Substitution of the alpha-70(Val) residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of alpha-70(Val) by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.     

Trapping a hydrazine reduction intermediate on the nitrogenase active site

A major challenge in understanding the mechanism of nitrogenase, the enzyme responsible for the biological fixation of N(2) to two ammonias, is to trap a nitrogenous substrate at the enzyme active site in a state that is amenable to further characterization. In the present work, a strategy is described that results in the trapping of the substrate hydrazine (H(2)N-NH(2)) as an adduct bound to the active site metal cluster of nitrogenase, and this bound adduct is characterized by EPR and ENDOR spectroscopies. Earlier work has been interpreted to indicate that nitrogenous (e.g., N(2) and hydrazine) as well as alkyne (e.g., acetylene) substrates can bind at a common FeS face of the FeMo-cofactor composed of Fe atoms 2, 3, 6, and 7. Substitution of alpha-70(Val) that resides over this FeS face by the smaller amino acid alanine was also previously shown to improve the affinity and reduction rate for hydrazine. We now show that when alpha-195(His), a putative proton donor near the active site, is substituted by glutamine in combination with substitution of alpha-70(Val) by alanine, and the resulting doubly substituted MoFe protein (alpha-70(Ala)/alpha-195(Gln)) is turned over with hydrazine as substrate, the FeMo-cofactor can be freeze-trapped in a S = (1)/(2) state in high yield ( approximately 70%). The presumed hydrazine-FeMo-cofactor adduct displays a rhombic EPR signal with g = [2.09, 2.01, 1.93]. The optimal pH for the population of this state was found to be 7.4. The EPR signal showed a Curie law temperature dependence similar to the resting state EPR signal. Mims pulsed ENDOR spectroscopy at 35 GHz using (15)N-labeled hydrazine reveals that the trapped intermediate incorporates a hydrazine-derived species bound to the FeMo-cofactor; in spectra taken at g(1) this species gives a single observed (15)N signal, A(g(1)) = 1.5 MHz.     

Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: effects of acetylene or ethylene on interactions with H+, HCN, and CN-

Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at alpha-195(His) (replaced by alpha-195(Asn) or alpha-195(Gln)) or at alpha-191(Gln) (replaced by alpha-191(Lys)), were used to probe the interactions of HCN and CN(-), both of which are present in NaCN solutions at pH 7.4, with nitrogenase. The first goal was to determine how added C(2)H(2) enhances the rate of CH(4) production from HCN reduction by wild-type nitrogenase. In the absence of C(2)H(2), wild-type Mo-nitrogenase showed a declining total electron flux, which is an overall measure of all products formed, as the NaCN concentration was increased from 1 to 5 mM, whereas the rates of both CH(4) and NH(3) production increased with increasing NaCN concentration. The NH(3) production rate exceeded the CH(4) production rate up to 5 mM NaCN, at which point they became equal. The "excess NH(3)" likely arises from the two-electron reduction of HCN to CH(2)=NH, some of which is released and hydrolyzed to HCHO plus NH(3). With added C(2)H(2), the rate of CH(4) production increased but only until it equaled that of NH(3) production, which remained unchanged. In addition, total electron flux was decreased even more at each NaCN concentration by C(2)H(2). The increased CH(4) production did not arise from the added C(2)H(2). The lowered total electron flux with C(2)H(2) present would decrease the affinity of the enzyme for HCN, making it a poorer competitor for the binding site. Thus, less CH(2)=NH would be displaced, more CH(2)=NH would undergo the full six-electron reduction, and the rate of CH(4) production would be enhanced. A second goal was to gain mechanistic insight into the roles of the amino acid residues in the alpha-subunit of the MoFe protein at positions alpha-191 and alpha-195 in substrate reduction. At 5 mM NaCN and in the presence of excess wild-type Fe protein, the specific activity for CH(4) production by the alpha-195(Asn), alpha-195(Gln), and alpha-191(Lys) MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the alpha-195(Asn) MoFe protein, total electron flux decreased with increasing NaCN concentration like wild type. However, the rates of both CH(4) and NH(3) production were maximal at 1 mM NaCN, and they remained unequal even at 5 mM NaCN. With the alpha-195(Gln) MoFe protein, the rates of production of both CH(4) and NH(3) were equal at all NaCN concentrations, and total electron flux was hardly affected by changing the NaCN concentration. With the alpha-191(Lys) MoFe protein, the rates of both CH(4) and NH(3) production were very low, but the rate of NH(3) production was higher, and both rates slowly increased with increasing NaCN concentration. A hypothesis, which is based on the varying apparent affinities of the altered MoFe proteins for HCN and CN(-), is advanced to explain the higher rate of NH(3) production versus the rate of CH(4) production and the effect of increasing NaCN concentration on electron flux to products. A new method for CH(3)NH(2) quantification showed that all four MoFe proteins produced CH(3)NH(2). Added CO significantly inhibited both CH(4) and NH(3) production from HCN with all MoFe proteins except for the alpha-191(Lys) MoFe protein, which still manifested its very low rate of NH(3) production but without CH(4) production. All of the MoFe proteins responded differently to the addition of C(2)H(2) to reactions containing NaCN. With the alpha-195(Asn) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production, but the rate of NH(3) production decreased much less. C(2)H(2) also exacerbated the inhibition of electron flux. With the alpha-195(Gln) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production substantially and about equally. C(2)H(2) also eliminated the slight decrease in total electron flux that was caused by NaCN. Added C(2)H(2) hardly affected the alpha-191(Lys) MoFe protein. (ABSTRACT TRUNCA     

Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C(2)H(2), C(2)H(4), CO, and H(2). The altered MoFe proteins used were the alpha-195(Asn) or alpha-195(Gln) MoFe proteins, which have either asparagine or glutamine substituting for alpha-histidine-195, and the alpha-191(Lys) MoFe protein, which has lysine substituting for alpha-glutamine-191. On the basis of K(m) determinations, C(2)H(2) was a particularly poor substrate for the nitrogenase containing the alpha-191(Lys) MoFe protein. Using C(2)D(2), a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C(2)D(2)H(2), and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the alpha-195(Asn) or the alpha-191(Lys) MoFe proteins, which also exhibited the highest rate of ethane production from C(2)H(2). These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C(2)H(4) was not a substrate of the nitrogenase with the alpha-191(Lys) MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the alpha-195(Gln) MoFe protein, both of which had a low V(max) and high K(m) (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the alpha-195(Asn) MoFe protein, which exhibited a K(m) of 48 kPa and a specific activity for C(2)H(6) formation from C(2)H(4) 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the alpha-195(Asn) MoFe protein produced cis-C(2)D(2)H(2) when turned over under trans-C(2)D(2)H(2). These results suggest that the C(2)H(4)-reduction site is affected by substitution at residue alpha-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C(2)H(4) flux-inhibition site is remote from the C(2)H(4)-reduction site. Added CO eliminated C(2)H(4) reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C(2)H(4) is not directly connected to C(2)H(4) reduction. Thus, C(2)H(4) has two binding sites, and the presence of CO affects only the site at which it binds as a substrate. When C(2)H(2) was added, it also eliminated C(2)H(6) production from C(2)H(4) and also did not relieve electron-flux inhibition fully. Thus, C(2)H(2) and C(2)H(4) are likely reduced at the same site on the MoFe protein. Two schemes are presented to integrate the results of the interactions of C(2)H(2) and C(2)H(4) with the MoFe proteins.     

Interaction of acetylene and cyanide with the resting state of nitrogenase alpha-96-substituted MoFe proteins.

The nitrogenase MoFe protein contains the active site metallocluster called FeMo-cofactor [7Fe-9S-Mo-homocitrate] that exhibits an S = 3/2 EPR signal in the resting state. No interaction with FeMo-cofactor is detected when either substrates or inhibitors are incubated with MoFe protein in the resting state. Rather, the detection of such interactions requires the incubation of the MoFe protein together with its obligate electron donor, called the Fe protein, and MgATP under turnover conditions. This indicates that a more reduced state of the MoFe protein is required to accommodate substrate or inhibitor interaction. In the present work, substitution of an arginine residue (alpha-96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to result in MoFe proteins that can interact with acetylene or cyanide in the as-isolated, resting state without the need for the Fe protein, or MgATP. The dithionite-reduced, resting states of the alpha-96(Leu)-, alpha-96(Gln)-, alpha-96(Ala)-, or alpha-96(His)-substituted MoFe proteins show an S = 3/2 EPR signal (g = 4.26, 3.67, 2.00) similar to that assigned to FeMo-cofactor in the wild-type MoFe protein. However, in contrast to the wild-type MoFe protein, the alpha-96-substituted MoFe proteins all exhibit changes in their EPR spectra upon incubation with acetylene or cyanide. The alpha-96(Leu)-substituted MoFe protein was representative of the other alpha-96-substituted MoFe proteins examined. The incubation of acetylene with the alpha-96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor signal with the appearance of a new EPR signal having inflections at g = 4.50 and 3.50. Incubation of cyanide with the alpha-96(Leu) MoFe protein also decreased the FeMo-cofactor EPR signal with concomitant appearance of a new EPR signal having an inflection at g = 4.06. The acetylene- and cyanide-dependent EPR signals observed for the alpha-96(Leu)-substituted MoFe protein were found to follow Curie law 1/T dependence, consistent with a ground-state transition as observed for FeMo-cofactor. The microwave power dependence of the EPR signal intensity is shifted to higher power for the acetylene- and cyanide-dependent signals, consistent with a change in the relaxation properties of the spin system of FeMo-cofactor. Finally, the alpha-96(Leu)-substituted MoFe protein incubated with (13)C-labeled cyanide displays a (13)C ENDOR signal with an isotropic hyperfine coupling of 0.42 MHz in Q-band Mims pulsed ENDOR spectra. This indicates the existence of some spin density on the cyanide, and thus suggests that the new component of the cyanide-dependent EPR signals arise from the direct bonding of cyanide to the FeMo-cofactor. These data indicate that both acetylene and cyanide are able to interact with FeMo-cofactor contained within the alpha-96-substituted MoFe proteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a consequence of both increased reactivity and accessibility of FeMo-cofactor under turnover conditions. We suggest that, for the wild-type MoFe protein, the alpha-96(Arg) side chain acts as a gatekeeper, moving during turnover in order to permit accessibility of acetylene or cyanide to a specific [4Fe-4S] face of FeMo-cofactor.     

Spectroscopic evidence for changes in the redox state of the nitrogenase P-cluster during turnover

Biological nitrogen fixation catalyzed by nitrogenase requires the participation of two component proteins called the Fe protein and the MoFe protein. Each alphabeta catalytic unit of the MoFe protein contains an [8Fe-7S] cluster and a [7Fe-9S-Mo-homocitrate] cluster, respectively designated the P-cluster and FeMo-cofactor. FeMo-cofactor is known to provide the site of substrate reduction whereas the P-cluster has been suggested to function in nitrogenase catalysis by providing an intermediate electron-transfer site. In the present work, evidence is presented for redox changes of the P-cluster during the nitrogenase catalytic cycle from examination of an altered MoFe protein that has the beta-subunit serine-188 residue substituted by cysteine. This residue was targeted for substitution because it provides a reversible redox-dependent ligand to one of the P-cluster Fe atoms. The altered beta-188(Cys) MoFe protein was found to reduce protons, acetylene, and nitrogen at rates approximately 30% of that supported by the wild-type MoFe protein. In the dithionite-reduced state, the beta-188(Cys) MoFe protein exhibited unusual electron paramagnetic resonance (EPR) signals arising from a mixed spin state system (S = 5/2, 1/2) that integrated to 0.6 spin/alphabeta-unit. These EPR signals were assigned to the P-cluster because they were also present in an apo-form of the beta-188(Cys) MoFe protein that does not contain FeMo-cofactor. Mediated voltammetry was used to show that the intensity of the EPR signals was maximal near -475 mV at pH 8.0 and that the P-cluster could be reversibly oxidized or reduced with concomitant loss in intensity of the EPR signals. A midpoint potential (Em) of -390 mV was approximated for the oxidized/resting state couple at pH 8.0, which was observed to be pH dependent. Finally, the EPR signals exhibited by the beta-188(Cys) MoFe protein greatly diminished in intensity under nitrogenase turnover conditions and reappeared to the original intensity when the MoFe protein returned to the resting state.     

Insights into substrate binding at FeMo-cofactor in nitrogenase from the structure of an α-70 MoFe protein variant

The X-ray crystal structure is presented for a nitrogenase MoFe protein where the alpha subunit residue at position 70 (α-70^Val) has been substituted by the amino acid isoleucine (α-70^Ile). Substitution of α-70^Val by α-70^Ile results in a MoFe protein that is hampered in its ability to reduce a range of substrates including acetylene and N₂, yet retains normal proton reduction activity. The 2.3 Å structure of the α-70^Ile MoFe protein is compared to the α-70^Val wild-type MoFe protein, revealing that the δ methyl group of α-70^Val is positioned over Fe6 within the active site FeMo-cofactor. This work provides strong crystallographic support for the previously proposed model that substrates bind and are reduced at a single 4Fe-4S face of the FeMo-cofactor and that when α-70^Val is substituted by α-70^Ile access of substrates to Fe6 of this face is effectively blocked. Furthermore the detailed examination of the structure provides the basis for understanding the ability to trap and characterize hydrides in the variant, contributing significantly to our understanding of substrate access and substrate reduction at the FeMo-cofactor active site of nitrogenase.     

Differential effects on N(2) binding and reduction, HD formation, and azide reduction with alpha-195(His)- and alpha-191(Gln)-substituted MoFe proteins of Azotobacter vinelandii nitrogenase

In contrast to the wild-type MoFe protein, neither the alpha-195(Asn) nor the alpha-191(Lys) MoFe protein catalyzed N(2) reduction to NH(3), when complemented with wild-type Fe protein. However, N(2) was bound by the alpha-195(Asn) MoFe protein and inhibited the reduction of both protons and C(2)H(2). The alpha-191(Lys) MoFe protein did not interact with N(2). With the alpha-195(Asn) MoFe protein, the N(2)-induced inhibition of substrate reduction was reversed by removing the N(2). Surprisingly, even though added H(2) also relieved N(2) inhibition of substrate reduction, the alpha-195(Asn) MoFe protein did not catalyze HD formation under a N(2)/D(2) atmosphere. This observation is the first indication that these two reactions have different chemical origins, prompting a revision of the current hypothesis that these two reactions are consequences of the same nitrogenase chemistry. A rationale that accounts for the dichotomy of the two reactions is presented. The two altered MoFe proteins also responded quite differently to azide. It was a poor substrate for both but, in addition, azide was an electron-flux inhibitor with the 195(Asn) MoFe protein. The observed reactivity changes are correlated with likely structural changes caused by the amino acid substitutions and provide important details about the interaction(s) of N(2,) H(2), D(2), and azide with Mo-nitrogenase.     

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Various S=3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alphaH195Q, alphaH195N, and alphaQ191K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N(2) with the alphaH195Q and alphaH195N variants, but not with the alphaQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alphaH195Q or alphaH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alphaQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze (57)Fe M?ssbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alphaH195Q and alphaH195N MoFe proteins can bind N(2), but alphaQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N(2) reduction.     

Mechanistic features and structure of the nitrogenase alpha-Gln195 MoFe protein

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [S?rlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.     

Molybdenum nitrogenase catalyzes the reduction and coupling of CO to form hydrocarbons

The molybdenum-dependent nitrogenase catalyzes the multi-electron reduction of protons and N(2) to yield H(2) and 2NH(3). It also catalyzes the reduction of a number of non-physiological doubly and triply bonded small molecules (e.g. C(2)H(2), N(2)O). Carbon monoxide (CO) is not reduced by the wild-type molybdenum nitrogenase but instead inhibits the reduction of all substrates catalyzed by nitrogenase except protons. Here, we report that when the nitrogenase MoFe protein α-Val(70) residue is substituted by alanine or glycine, the resulting variant proteins will catalyze the reduction and coupling of CO to form methane (CH(4)), ethane (C(2)H(6)), ethylene (C(2)H(4)), propene (C(3)H(6)), and propane (C(3)H(8)). The rates and ratios of hydrocarbon production from CO can be adjusted by changing the flux of electrons through nitrogenase, by substitution of other amino acids located near the FeMo-cofactor, or by changing the partial pressure of CO. Increasing the partial pressure of CO shifted the product ratio in favor of the longer chain alkanes and alkenes. The implications of these findings in understanding the nitrogenase mechanism and the relationship to Fischer-Tropsch production of hydrocarbons from CO are discussed.     

Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster.

The Azotobacter vinelandii genes encoding the nitrogenase structural components are clustered and ordered: nifH (Fe protein)-nifD (MoFe protein alpha subunit)-nifK (MoFe protein beta subunit). In this study various A. vinelandii mutant strains which contain defined deletions within the nitrogenase structural genes were isolated and studied. Mutants deleted for the nifD or nifK genes were still able to accumulate significant amounts of the unaltered MoFe protein subunit as well as active Fe protein. Extracts of such nifD or nifK deletion strains had no MoFe protein activity. However, active MoFe protein could be reconstituted by mixing extracts of the mutant strains. These results establish an approach for the purification of the individual MoFe protein subunits. Mutants lacking either or both of the MoFe protein subunits were still able to synthesize the iron-molybdenum cofactor (FeMo-cofactor), indicating that in A. vinelandii the FeMo-cofactor is preassembled and inserted into the MoFe protein. In contrast, a mutant strain lacking both the Fe protein and the MoFe protein failed to accumulate any detectable FeMo-cofactor. The further utility of specifically altered A. vinelandii strains for the study of the assembly, structure, and reactivity of nitrogenase is discussed.     

Effects of substrates (methyl isocyanide,C2H2) and inhibitor (CO) on resting-state wild-type and NifV(-)Klebsiella pneumoniae MoFe proteins

We report the use of electron nuclear double resonance (ENDOR) spectroscopy to examine how the metal sites in the FeMo-cofactor cluster of the resting nitrogenase MoFe protein respond to addition of the substrates acetylene and methyl isocyanide and the inhibitor carbon monoxide. 1H, 57Fe and 95Mo ENDOR measurements were performed on the wild-type and the NifV(-)proteins from Klebsiella pneumoniae. Among the molecules tested, only the addition of acetylene to either protein induced widespread changes in the 57Fe ENDOR spectra. Acetylene also induced increases in intensity from unresolved protons in the proton ENDOR spectra. Thus we conclude that acetylene may bind to the resting-state MoFe protein to perturb the FeMo-cofactor environment. On the other hand, the present results show that methyl isocyanide and carbon monoxide do not substantially alter the FeMo cofactor's geometric and electronic structures. We interpret this as lack of interaction between those two molecules and the FeMo cofactor in the resting state MoFe protein. Thus, although it is generally accepted that substrates or inhibitors bind to the FeMo-cofactor only under turnover condition, this work provides evidence that at least one substrate can perturb the active site of nitrogenase under non-catalytic conditions.     

Electron transfer within nitrogenase: evidence for a deficit-spending mechanism

The reduction of substrates catalyzed by nitrogenase 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 through these three metalloclusters 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. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188(Ser) residue substituted by cysteine and the other having the β-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).     

EXAFS studies on the PN and POX states of the P-clusters in nitrogenase

The MoFe protein of nitrogenase is an α₂β₂ tetramer that contains two each of two different types of metal centers, the FeMo-cofactor and the P-clusters. The function of the P-clusters is believed to be to accept electrons from the Fe protein of nitrogenase and to donate them to the FeMo-cofactor. We have studied the P-clusters of Azotobacter vinelandii nitrogenase in both the P^N and P^OX states utilizing Fe K-edge X-ray absorption spectroscopy. Since the MoFe holoprotein contains the seven iron FeMo-cofactor centers in addition to P-clusters, we have utilized a FeMo-cofactor-deficient MoFe protein synthesized by the Δnif H strain DJ54. That MoFe protein is an α₂β₂ tetramer that contains P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the P^OX state. Several important results have emerged from our XAS studies. The first shell Fe-S coordination shows the same average Fe-S distance (2.26?Å) in both states. The second coordination shells could only be well fit using two different Fe-Fe contributions. In both states, short Fe-Fe components with distances of 2.57?Å and 2.42?Å for the P^N and P^OX states, respectively, were required to complement longer 2.75?Å and 2.70?Å distances. Understanding of the P-cluster structure is essential if we are to make advances in understanding the role of the P-clusters and their participation in electron transfer through the nitrogenase system.     

Cyanamide: a new substrate for nitrogenase

(1) Cyanamide (N identical to C-NH2) has been shown to be a substrate for purified Mo-nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum, with apparent Km values near 0.8 mM. (2) Reduction products were CH4, CH3NH2 and NH3 formed by pathways requiring 6 or 8 electrons: N identical to CNH2 + 6e + 6H+----CH3NH2 + NH3; N identical to CNH2 + 8e + 8H+----CH4 + 2NH3 (3) Acetylene reduction and hydrogen evolution were inhibited more than 75% by cyanamide (10 mM). Cyanamide also inhibited total electron flux at nitrogenase protein component ratios (Fe/MoFe) near 10. (4) Cyanamide was also a substrate for the recently isolated Va-nitrogenase of A. chroococcum, but with an apparent Km of 2.6 mM showed weaker binding and an 8-fold lower Vmax than did either Mo-nitrogenase. (5) The component ratios of nitrogenase proteins favouring CH4 formation was 3.5 Fe/MoFe protein and 1 Fe/VaFe protein.     

Isolation and characterization of an acetylene-resistant nitrogenase

A genetic strategy was developed for the isolation of a mutant strain of Azotobacter vinelandii that exhibits in vivo nitrogenase activity resistant to inhibition by acetylene. Examination of the kinetic features of the altered nitrogenase MoFe protein produced by this strain, which has serine substituted for the alpha-subunit Gly(69) residue, is consistent with other studies that indicate the MoFe protein normally contains at least two acetylene binding/reduction sites. The first of these is a high affinity site and is the one primarily accessed during typical acetylene reduction assays. Results of the present work indicate that this acetylene binding/reduction site is not directly relevant to the mechanism of nitrogen reduction because it can be eliminated or severely altered without significantly affecting nitrogen reduction. Elimination of this site also results in the manifestation of a low affinity acetylene-binding site to which both acetylene and nitrogen are able to bind with approximately the same affinity. In contrast to the normal enzyme, nitrogen and acetylene binding to the altered MoFe protein are mutually competitive. The location of the alpha-Ser(69) substitution is interpreted to indicate that the 4Fe-4S face of the FeMo cofactor capped by the alpha-subunit Val(70) residue is the most likely region within FeMo cofactor to which acetylene binds with high affinity.