Relationship Between nifZ and the Synthesis of P cluster in Nitrogenase MoFe Protein of Azotobacter vinelandii

In comparison with OP MoFe protein from wild type strain Azotobacter vinelandii Lipmann, the C2H2-reduction activity and atom ratio of Fe to Mo of △nifZ MoFe protein from a nifZ deletion strain of A. vinelandii were remarkably decreased. FeMoco, which were extracted from these two proteins under the same condition, were almost similar to each other in activity and metal composition, and the circular dichroism (CD) spectra of these proteins were significantly different from each other. In the visible region except 540 750 nm, the △ε at 380 - 540 nm of △nifZ MoFe protein decreased and had a peculiar sharp negative peak around 430 nm; and in the ultraviolet region, the peaks at 208 nm and 222 nm were higher than those of OP MoFe protein. △nifZ MoFe protein could be crystallized in a suitable concentration of PEG 8000 and MgCl2, the size of crystals and amount of precipitation seemed to be related to the above-mentioned negative peaks. The results showed that △nifZ of Azotobacter vinelanclii might be related to the synthesis of P-cluster, rather than to that of FeMoco, which resulted in its conformation, stability and process of crystallization.     

Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.     

Studies on Assembly of Vanadium-iron Protein in Vitro

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with ophenanthroline under anaerobic or aerobic condition,inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive MoFe protein with a reconstituent solution containing NaVO3,ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The proton reduction activity and absorption spectrum of the reconstituted protein could be well restored, but its C2H2-reduction activity could not be recovered. Its CD spectrum could be recovered except for the 550nm to 650nm region which differed from that of the reduced MoFe protein. The results showed that the reconstituted protein was different from MoFe protein,but was similar to vanadium-iron protein.     

含铼重组液对部分缺失金属原子簇的钼铁蛋白的激活作用

棕色固氮菌(AzotobactervinelandiiLipmann)固氮酶MoFe蛋白经邻菲啉和空气处理后,成为部分缺失P_cluster和FeMoco的失活蛋白。与由Re2O7、高柠檬酸铁、Na2S和二硫苏糖醇(DTT)组成的无圆二色(CD)谱信号的重组液保温后,保温蛋白对乙炔和质子还原的活性都得以显著恢复;紫外和可见光CD谱虽有明显恢复,但仍与还原MoFe蛋白有所差异。这表明:1)保温的蛋白液中除含有未被邻菲啉等处理而破坏的完整MoFe蛋白外,还可能存在新组装的含Re的固氮酶;2)新组装的ReFe蛋白和MoFe蛋白可能在固氮能力上相似,而在结构上有所差别     

Studies on the Reactivation of Aerated Nitrogenase MoFe Protein from Azotobacter vinelandii by the Compounds of iron Molybdenum and Sulfur

After the exposure to air, the crystalline nitrogenase MoFe protein from Azotobacter vinelandii was resulted in the remarkable increase in its absorption (ABS) and the significant decrease in its activity and circular dichroism (CD). However, when the aerated MoFe protein was incubated with the reconstituting solution which consisted of Na2MoO4, ferric citrate, Na2S and dithiothreitol, the ABS and CD of the aerated. MoFe protein both were completely restored, simultaneously with the significant restoration of acetylene reduction. It is shown that the P-cluster and other parts related to the protein activity which was damaged by O2 are able to be repaired to a certain extent by the reconstituting solution.     

The Effect of Manganese on the Reconstitution of Partial Metallocluster-deficient Nitrogenase Molybdenum-Iron Protein

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with O-phenanthroline (O-phen) and O2, inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive protein with a reconstituent solution containing KMnO4, ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The absorption spectrum and C2H2, H+ and N2 reduction activity of the reconstituted protein could be well restored to the state of the reduced MoFe protein. However, the α-helix and CD spectrum at 380—550 nm and at 620—670 nm of the reconstituted protein were somewhat different from those of the reduced MoFe protein. The results showed that: (1) the reconstituted protein was composed of the assembled protein which might be a MnFe protein due to the reconstitution of the metalloclusterdeficient MoFe protein with Mn-containing solution and MoFe protein in which metalloclusters were still intact after the treatment with O-phen and O2; (2) It might be possible that the MnFe protein and MoFe protein were similar in the ability of nitrogen fixation, but were somewhat different in the structure from each other.     

Reactivation of Partially Metallocluster deficient MoFe Protein by Rhenium-containing Reconstituent Solution

When the reduced MoFe protein from Azotobacter vinelandii Lipmann was treated with ophenanthroline and air, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. After incubating the treated protein with a reconstituent solution containing Re2OT, ferric homocitrate, Na2S and dithiothreitol, which had no circular dichroism (CD) signal, the ultraviolet and visible CD spectra, the C2H2 and H+ -reduction activity of the incubated protein were significantly recovered. However, the spectra were somewhat different from those of the reduced MoFe protein. The results showed that: 1) in the incubated protein solution there was possibly a new recombined ReFe protein besides the intact MoFe protein which was not destroyed by the treatment with o-phenanthroline and air; 2) it might be possible that both ReFe protein and MoFe protein exhibited similar ability of nitrogen fixation, although they were somewhat different in structure.     

Structural Requirement for Clusters to be Reconstituted with the FeMoCo-Deficient Molybdenum-Iron Protein

By incubating the reduced MoFe protein from Azotobacter vinelandii with O-phenanthroline under air and chromatographying the incubated solution on Sephadex G-25 column, inactive MoFe protein could be obtained. Its acetylene-reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO4, ferric homoeitrate, Na2S and dithiothreitol, but also with a mixture of 4Fe : 4S clusters and another cluster which had two structure units of 1Mo : 3Fe : 4S-bridged by three -OCH3 at the Mo atoms. Neither the reconstituent solution nor the mixture could reactivate apo-MoFe proteins from the mutants deleting nile and nifH genes and from the mutant UW45, which could be reactivated by the FeMoeo extracted from the MoFe protein. The results indicated that the FeMoeo-defieient MoFe proteins from these mutants seemed to be reconstituted only by the clusters which were probably structures only similar to FeMoeo. The partially metalloeluster-deficient MoFe protein could be reconstituted by the clusters with a certain kind of structure and composition; and was changed into different nitrogenase proteins with the ability to fix nitrogen.     

Purification and Properties of Nitrogenase MoFe Protein from a nif Z Deletion Strain of Azotobacter vinelandii

The MoFe protein of the nif Z deletion strain (△nif Z MoFe protein) of Azotobac ter vinelandii designated DJ 194 was purified and some properties were studied. The cell free extract of DJ 194 was more sensitive to O2 and heat than the wild-type extract. The specific activity of the purified DJ 194 protein was 283 nmol C2H2 reduced/(min · mg protein), which was much lower than that of purified wild-type A. vinelandii MoFe protein. The △nif Z MoFe protein exhibited a visible similar absorption spectra as the wild type MoFe protein, yet showed significant difference in CD and MCD spectra at the region about 450 mm com paring with the spectral property of the wild-type MoFe protein. This seems to indicate that the P-cluster of the △nif Z MoFe protein was modified, which might be the cause of the low activity of the DJ 194 MoFe protein.     

钼铁蛋白铁钼辅因子的有机组分对其功能的影响

棕色固氮菌(Azotobacter vinelandii)固氮酶的钼铁蛋白经邻菲啰啉在厌氧或有氧环境中处理后,变为 P-cluster 单一缺失或 P-cluster 和 FeMoco 同时缺失的失活钼铁蛋白。含柠檬酸盐或高柠檬酸盐的重组液都使这两种失活蛋白能恢复固氮酶重组的 H~ 和 C_2H_2还原活性,活性恢复程度随反映钼铁蛋白中金属原子簇含量变化的圆二色和磁圆二色谱及金属含量的恢复程度的提高而提高,但它们固 N_2能力的恢复程度则不相同:P-cluster 单一缺失的蛋白用两种重组液重组后均可恢复其固 N_2能力,而 P-cluster 和 FeMoco 同时缺失的蛋白,只有用含高柠檬酸盐的重组液重组才恢复其固 N_2能力,表明含不同有机组分的重组液所组装的 P-cluster 均与天然状态相同,只有含高柠檬酸盐的重组液所组装的 FeMoco 才与天然状态相同,从而证明高柠檬酸盐是 FeMoco 的必需的有机组分。     

Reactivation of Partially Metallocluster-deficient MoFe Protein by Chromium-Containing Reconstituent Solution

By treating the reduced MoFe protein from Azotobacter vinelandii with o-phenanthroIi e and O2, partially deficient in both FeMoco and P-cluster and inactive protein could be o rained. After incubating the treated protein with a reconstituent solution containing K2CrO4, ferric homocitrate, Na2S and dithiothreitol, a reactivated protein could be obtained. The absorption spectrum, circular dichroism spectrum, and the C2H2 and proton reduction activities of the reactivated protein were remarkably recovered. However, the spectra were somewhat different from those of the reduced MoFe protein. The results showed that some of the reactivated protein might be Cr-containing protein (CrFe protein) which were similar in function, but somewhat different in structure from MoFe protein.     

含铬重组液激活部分缺失金属原子簇的钼铁蛋白的研究

棕色固氮菌（Ａｚｏｔｏｂａｃｔｅｒ ｖｉｎｅｌａｎｄｉｉ）固氮酶钼铁蛋白经邻菲口罗啉和Ｏ２ 处理后,变为部分缺失ＦｅＭｏｃｏ 和Ｐ－ｃｌｕｓｔｅｒ的失活蛋白。与由Ｋ２ＣｒＯ４、高柠檬酸铁、Ｎａ２Ｓ和二硫苏糖醇组成的重组液保温后,处理蛋白对乙炔和质子还原的活性都得以显著恢复;然而,它的吸收光谱和圆二色谱虽有明显恢复,但仍与还原钼铁蛋白有所不同。这表明,激活蛋白中也许存在功能与钼铁蛋白相似,而结构则有所差异的含铬（ＣｒＦｅ）蛋白     

Complex formation and O2 sensitivity of Azotobacter vinelandii nitrogenase and its component proteins

The O2 stability of the MoFe protein, the Fe protein, a 1:1 mixture of these proteins, and a 1:1 mixture in the presence of the Azotobacter vinelandii FeS-II protein has been studied as a function of time under controlled O2 partial pressures. The Fe protein is much more sensitive to O2 exposure than is the MoFe protein. The presence of the FeS-II protein at a 1:1 ratio with the component proteins measurably increases the O2 stability of the MoFe and Fe proteins. O2 inactivation of the MoFe protein was studied in some detail and found to be quite complex. At least three partially overlapping reactions are suggested. The first is the reversible oxidation of the metal clusters of the MoFe protein to the combined extent of 12 electrons with full retention of activity. The second phase consists primarily of activity loss with little increase in the extent of reversible oxidation. The third phase continues to decrease the protein activity but is also accompanied by formation of a g = 2.0 EPR signal and more extensive oxidation. Ultracentrifugation studies of the FeS-II protein at a 1:1:1 ratio with the Fe and MoFe proteins do not support the formation of the Bulen complex. The formation of other O2-stable complexes is discussed.     

Comparative Studies on Mn,Cr and Mo Contained Reconstituent Solutions and Proteins Reconstituted with Partially Metallocluster-deficient MoFe Protein

By treating the reduced MoFe protein from Azotobacter vinelandii with o-phenanthroline and O2, an inactive protein partially deficient in both FeMoco and P-cluster could be obtained. The inactive protein could be reactivated by the reconsfituent solutions of different colours which were prepared from mixture in different proportions of ferric homocitrate, Na2S and dithiothreitol (DTT) with K2CrO4, KMnO4 and Na2MoO4, respectively, the inactive protein could be somewhat reactivated by DTF but not by the other compounds or their mixtures which were deficient in one or two of the above compounds. 99Mo was found in the reconstituted protein of the inactive MoFe protein with a reconstituent solution containing 99Mo. Its measurement of differential perturbed angular correlation indicated that 41% of 99Mo in the reconstituted protein were in a state similar to that of 99Mo in 99MoFe protein from K. pneumoniae. The results showed that the reactivation of the inactive MoFe rotein was mainly based on the restoration of its metallocluster content. It seemed to be reasonable to postulate that Mn- or Cr-containing nitrogenase could be obtained likewise by the reconstitution of the inactive protein with the Mn- or Cr-containing reconstituent solution.     

钼,铁,硫化合物激活曝氧的棕色固氮菌固氮酶钼铁蛋白的研究

曝氧后,棕色固氮菌(Azotobacter vinelandii)固氮酶钼铁蛋白的催化活性和圆二色信号都显著降低,而吸收光谱则显著增加。与钼、铁、硫化合物和二硫苏糖醇组成的重组溶液保温后,曝氢蛋白的圆二色信号和吸收光谱几乎完全恢复至天然状态的同时,乙炔还原活性也得到了显著的恢复,表明重组溶液可使曝氧蛋白中的 P-cluster和其它活性部位都得到了不同程度的修复。     

Evidence for the selective population of FeMo cofactor sites in MoFe protein and its molecular recognition by the Fe protein in transition state complex analogues of nitrogenase

We have collected synchrotron x-ray solution scattering data for the MoFe protein of Klebsiella pneumoniae nitrogenase and show that the molecular conformation of the protein that contains only one molybdenum per alpha(2)beta(2) tetramer is different from that of the protein that has full occupancy i.e. two molybdenums per molecule. This structural finding is consistent with the existence of MoFe protein molecules that contain only one FeMo cofactor site occupied and provides a rationale for the 50% loss of the specific activity of such preparations. A stable inactive transition state complex has been shown to form in the presence of MgADP and AlF(4)(-). Gel filtration chromatography data show that the MoFe protein lacking a full complement of the cofactor forms initially a 1:1 complex before forming a low affinity 1:2 complex. A similar behavior is found for the MoFe protein with both cofactors occupied, but the high affinity 1:2 complex is formed at a lower ratio of Fe protein/MoFe protein. The 1:1 complex, MoFe protein-Fe protein x (ADP x AlF(4)(-))(2), formed with MoFe protein that lacks one of the cofactors, is stable. X-ray scattering studies of this complex have enabled us to obtain its low resolution structure at approximately 20-A resolution, which confirms the gel filtration finding that only one molecule of the Fe protein binds the MoFe protein. By comparison with the low resolution structure of purified MoFe protein that contains only one molybdenum per tetramer, we deduce that the Fe protein interacts with the FeMo cofactor-binding alpha-subunit of the MoFe protein. This observation demonstrates that the conformation of the alpha-subunit or the alpha beta subunit pair that lacks the FeMo cofactor is altered and that the change is recognized by the Fe protein. The structure of the 1:1 complex reveals a similar change in the conformation of the Fe protein as has been observed in the low resolution scattering mask and the high resolution crystallographic study of the 1:2 complex where both cofactors are occupied and with the Fe protein bound to both subunits. This extensive conformational change observed for the Fe protein in the complexes is, however, not observed when MgATP or MgADP binds to the isolated Fe protein. Thus, the large scale conformational change of the Fe protein is associated with the complex formation of the two proteins.     

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes.

Circular dichroism (CD) spectra were measured for cytochromes P-450 (P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in alpha-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45--50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250--310 nm) and weaker negative CD spectra associated with Soret transitions for PBP-448 ([theta] = 50 000) and MCP-448 ([theta] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.     

The role of the MoFe protein alpha-125Phe and beta-125Phe residues in Azotobacter vinelandii MoFe protein-Fe protein interaction

Site-directed mutagenesis and gene-replacement techniques were used to substitute alanine for the MoFe protein alpha- and beta-subunit phenylalanine-125 residues both separately and in combination. These residues are located on the surface of the MoFe protein near the pseudosymmetric axis of symmetry between the alpha- and beta-subunits. Altered MoFe proteins that contain an alanine substitution at only one of the respective positions exhibit proton reduction activities of about 25-50% when compared to that of the wild-type protein. The lower level of proton reduction also corresponds with decreases in the rates of MgATP hydrolysis. The MoFe protein which contains alanine substitutions in both the alpha- and beta- subunits did not exhibit any proton reduction activity or MgATP hydrolysis. Stopped flow spectrophotometry of the singly substituted MoFe proteins indicate primary electron transfer rate constants approximately an order of magnitude slower than what is observed for wild-type MoFe protein, while no primary electron transfer is observed for the doubly substituted MoFe protein. The doubly substituted MoFe protein is able to interact with the Fe protein as shown by chemical crosslinking experiments. However, this protein does not form a tight complex with the Fe protein when treated with MgADP-AlF4- or when using the altered 127delta Fe protein. Stopped flow spectrophotometry was also used to quantitate the first-order dissociation rate constants for the two component proteins. These results suggest that the 125Phe residues are involved in an early event(s) that occurs upon component protein docking and could be involved in eliciting MgATP hydrolysis.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

Mechanistic interpretation of the dilution effect for Azotobacter vinelandii and Clostridium pasteurianum nitrogenase catalysis

Nitrogenase activity for Clostridium pasteurianum (Cp) at a Cp2:Cp1 ratio of 1.0 and Azotobacter vinelandii (Av) at Av2:Av1 protein ratios (R) of 1, 4 and 10 is determined as a function of increasing MoFe protein concentration from 0.01 to 5 microM. The rates of ethylene and hydrogen evolution for these ratios and concentrations were measured to determine the effect of extreme dilution on nitrogenase activity. The experimental results show three distinct types of kinetic behavior: (1) a finite intercept along the concentration axis (approximately 0.05 microM MoFe); (2) a non-linear increase in the rate of product formation with increasing protein concentration (approximately 0.2 microM MoFe) and (3) a limiting linear rate of product formation at high protein concentrations (>0.4 microM MoFe). The data are fitted using the following rate equation derived from a mechanism for which two Fe proteins interact cooperatively with a single half of the MoFe protein. (see equation) The equation predicts that the cubic dependence in MoFe protein gives rise to the non-linear rate of product formation (the dilution effect) at very low MoFe protein concentrations. The equation also predicts that the rate will vary linearly at high MoFe protein concentrations with increasing MoFe protein concentration. That these limiting predictions are in accord with the experimental results suggests that either two Fe proteins interact cooperatively with a single half of the MoFe protein, or that the rate constants in the Thorneley and Lowe model are more dependent upon the redox state of MoFe protein than previously suspected [R.N. Thornley and D. J. Lowe, Biochem. J. 224 (1984) 887-894]. Previous Klebsiella pneumoniae and Azotobacter chroococcum dilution results were reanalyzed using the above equation. Results from all of these nitrogenases are consistent and suggest that cooperativity is a fundamental kinetic aspect of nitrogenase catalysis.