ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTInvestigation of CO bound to inhibited forms of nitrogenase MoFe protein by 13C ENDORRobert C. Pollock, Hong-In Lee, Linda M. Cameron, Victoria J. DeRose, Brian J. Hales, W. H. Orme-Johnson, and Brian M. HoffmanCite this: J. Am. Chem. Soc. 1995, 117, 33, 8686–8687Publication Date (Print):August 1, 1995Publication History Published online1 May 2002Published inissue 1 August 1995https://pubs.acs.org/doi/10.1021/ja00138a033https://doi.org/10.1021/ja00138a033research-articleACS PublicationsRequest reuse permissionsArticle Views342Altmetric-Citations89LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The identification of calcium-binding proteins in urine and kidney stones has led to a closer look at the role of matrix proteins in urolithiasis. We analyzed five struvite stones for protein content and identified two bands (8 and 14 KDa) that were confirmed by gel electrophoresis and amino acid sequencing to be calgranulin. This protein, which is known by several other names, has bacteriostatic antifungal activity. Its role in the formation of struvite stones warrants further investigation.

Mg-ATP binds to the iron protein component of nitrogenase. The magnetic field dependence of the linear electric field effect (LEFE) in pulsed EPR is consistent with a single 4Fe-4S cluster. The LEFE is virtually unaltered when Mg-ATP is bound. Electron spin echo envelope modulation techniques were employed to evaluate the possibility of a magnetic interaction between 31P of Mg-ATP and the Fe-S center of the iron protein. None was detected. However, weak modulations possibly attributable to peptide 14N were seen, and these were slightly shifted by Mg-ATP addition. Further, protons in the vicinity of the Fe-S cluster of the protein readily exchange with D2O, and this process is unaffected by Mg-ATP.

We have determined the complete nucleotide sequences of three functionally related nitrogen assimilation regulatory genes from Klebsiella pneumoniae and Rhizobium meliloti. These genes are: 1) The K. pneumoniae general nitrogen assimilation regulatory gene ntrC (formerly called glnG), 2) the K. pneumoniae nif-specific regulatory gene nifA, and 3) an R. meliloti nif-specific regulatory gene that appears to be functionally analogous to the K. pneumoniae nifA gene. In addition to the DNA sequence data, gel-purified K. pneumoniae nifA protein was used to determine the amino acid composition of the nifA protein. The K. pneumoniae ntrC and nifA genes code for proteins of 52,259 and 53,319 d respectively. The R. meliloti nifA gene codes for a 59,968 d protein. A central region within each polypeptide, consisting of approximately 200 amino acids, is between 52% and 58% conserved among the three proteins. Neither the amino termini nor the carboxy termini show any conserved sequences. Together with data that shows that the three regulatory proteins activate promoters that share a common consensus sequence in the -10 (5'-TTGCA-3') and -23 (5'-CTGG-3') regions, the sequence data presented here suggest a common evolutionary origin for the three regulatory genes.

Abstract Chemical and physical analyses indicate that the ironmolybdenum cofactor (FeMo(co)) of nitrogenase contains 6–8 mol or iron and 4–6 mol of sulfur per mol of molybdenum. The physical properties of this cofactor suggest that it contains a novel MoFeS cluster. Extended X-ray absorption fine structure (EXAFS) data taken at the Mo edge indicate that the molybdenum has two or three iron atoms and four or five sulfur atoms as nearest neighbors. Several models are consistent with these data. More information concerning the iron environment is needed to better define the structure of the FeMo(co). In this talk, we will present our recent results [1] on the iron edge EXAFS of the FeMo(co) from Azotobacter vinelandii and relate structural information about the iron sites in the cluster. In a related study, we have obtained the iron K-edge EXAFS of the 3Fe ferredoxin II of Desulfovibrio gigas in the oxidized and reduced states [2]. For both states, interpretation of the EXAFS data suggests that the FeS distance is near 2.25 A, in agreement with crystallographic studies of model compounds and proteins containing 2Fe2S and 4Fe4S centers, as well as with a recent crystallographic study of Azotobacter vinelandii ferredoxin I (D. Ghosh, W. Furey, Jr., S. O'Donnell and C.D. Stout, J. Biol. Chem. 256 , 4185 (1981).). The FeFe distance of 2.7 A, however, agrees with similar distances observed in other FeS centers, but disagrees with the 3Fe cluster in the Azotobacter vinelandii ferredoxin I structure, for which an FeFe distance of 4.2 A was reported. We conclude that the two 3Fe ferredoxins may have substantially different core dimensions, a possibility apparently unique to 3Fe centers among known FeS systems in proteins. The implication of such structural variation of 3Fe centers in the nitrogenase problem will be discussed.

We describe a method for the differentiation of 3iron from 2-iron and 4-iron Fe/S proteins based on consideration of both the magnetic field dependence of shifts in g induced by an externally applied electric field (LEFE) and the continuous wave EPR spectra properties.The magnetic field dependence and the magnitude of the LEFE for 3-iron ferredoxins are similar to those for 4-iron ferredoxins but differ considerably from those for 2-iron ferredoxins or for high potential iron proteins.Furthermore, as 3-iron ferredoxins and high potential iron proteins are EPR-active when oxidized while 2-iron and 4-iron ferredoxins are only EPR-active when reduced, the differentiation among all of them can be made on the basis of both continuous wave EPR and LEFE properties, but not by each individually.

We have obtained the iron K-edge extended X-ray adsorption fine structure spectra of the 3Fe ferredoxin II of Desulfovibrio gigas in the oxidized and reduced states. For both states, interpretation of the EXAFS data suggests that the Fe-S first shell coordination distance is near 2.25 A, in agreement with crystallographic studies of model compounds and proteins containing 2Fe-2S and 4Fe-4S centers, as well as with a recent crystallographic study of Azotobacter vinelandii ferredoxin I (Ghosh, D., Furey, W., Jr., O'Donnell, S., and Stout, C. D. (1981) J. Biol. Chem. 256, 4185-4192). The apparent Fe-Fe distance we obtain for the desulfovibrio protein (2.7 A) also agrees with similar distances seen in other Fe-S centers, except with the 3Fe cluster in the Azotobacter vinelandii ferredoxin I structure, for which an Fe-Fe distance of 4.2 A was reported. We conclude that either the two 3Fe ferredoxins have substantially different core dimensions, a possibility apparently unique to 3Fe centers among known Fe-S systems in proteins, or that one (or more) of the structural studies is in substantial error.

The difference between the magnetic susceptibilities of native and thionine-oxidized molybdenum-iron protein from Azotobacter vinelandii nitrogenase was measured by the nuclear magnetic resonance method. Reversible oxidation of the MoFe protein by 4 to 8 electron eq of thionine/mol made the protein more paramagnetic than it was in the native state. The NMR susceptibility results were analyzed in terms of a model for the spin states of the iron centers in the MoFe protein based on low temperature electron paramagnetic resonance and Mössbauer spectral studies. The model proposes that the native protein contains 2 "M" centers (S = 3/2) and 4 "P" centers (S = 0)/mol and that the oxidized protein has diamagnetic M centers and paramagnetic P centers with S greater than or equal to 3/2. Assuming that this model holds at 280 K, the NMR susceptibility results show that the effective magnetic moment of the oxidized P centers is larger than that of the native M centers. Based on an analysis in terms of spin only magnetic moments, the susceptibility results suggest that the P centers in the oxidized protein are S = 5/2 systems.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIron-57 ENDOR of the nitrogenase molybdenum-iron proteinBrian M. Hoffman, Ronald A. Venters, James E. Roberts, Mark Nelson, and W. H. Orme-JohnsonCite this: J. Am. Chem. Soc. 1982, 104, 17, 4711–4712Publication Date (Print):August 1, 1982Publication History Published online1 May 2002Published inissue 1 August 1982https://pubs.acs.org/doi/10.1021/ja00381a050https://doi.org/10.1021/ja00381a050research-articleACS PublicationsRequest reuse permissionsArticle Views111Altmetric-Citations43LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRenal mitochondrial ferredoxin active in 25-hydroxyvitamin D3 1.alpha.-hydroxylase. Characterization of the iron-sulfur cluster using interprotein cluster transfer and electron paramagnetic resonance spectroscopyPoksyn S. Yoon, J. Rawlings, W. H. Orme-Johnson, and H. F. DeLucaCite this: Biochemistry 1980, 19, 10, 2172–2176Publication Date (Print):May 13, 1980Publication History Published online1 May 2002Published inissue 13 May 1980https://pubs.acs.org/doi/10.1021/bi00551a027https://doi.org/10.1021/bi00551a027research-articleACS PublicationsRequest reuse permissionsArticle Views41Altmetric-Citations20LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

We have studied the high and low potential ironsulfur centers of an Azotobacter vinelandii ferredoxin with Mossbauer spectroscopy.The data suggest that the high potential cluster is of the Fe4Sc type and that the low potential center (Eo = -420 mV) represents a novel type of cluster.The oxidized low potential center which exhibits an isotropic EPR signal at g = 2.01 has at least 2 distinct iron sites with identical quadrupole splittings, AEQ = 0.63 mm/s, and isomeric shifts, B = 0.27 mm/s, suggesting high spin ferric ions with a tetrahedral sulfur ligation.The two sites display dras-.tically different magnetic hyperfine interactions.Studies in strong applied magnetic fields show that the magnetic hyperfine coupling constants have opposite signs, a strong indication of spin-coupling.Upon reduction, the g = 2.01 center is found to have an electronic ground state of integer, nonzero spin, Le. the center is paramagnetic.The Mossbauer data force us to consider the possibility that the low potential center might contain 3 iron atoms, a suggestion compatible with the data obtained for the oxidized center.Since 2 high spin ( S = 5/2) ferric ions cannot be coupled to yield the halfintegral spin observed for the oxidized center, a third, half-integral spin is required.If this spin is not furnished by a 3rd iron atom, as suggested by the data obtained for the reduced center, a ligand radical should be considered.Data reported in the accompanying paper (Stout, C. D., Ghosh, D., Pattabhi, V., and Robbins, A. H. (1980) J BioL Chem 266,1797-1800) support the concept of a 3-Fe center, as well as the presence of a well defined Fe4S4 cluster in this protein.Cluster displacement experiments reveal that FeaSr and Fe4S4 cores can be transferred from this ferredoxin into appropriate protein acceptors.These experiments do not rule out the presence of a trinuclear iron cluster.

The production of deuterated methane by Methanobacterium thermoautotrophicum in H2O-D2O mixtures was examined by high-resolution mass spectrometry. The hydrogen in the methane arose solely from water and not from hydrogen gas. Hydrogen gas served only as an electron source in methanogenesis. A whole-cell product isotope discrimination of 1.5 favoring hydrogen over deuterium was observed in methane production in 81 atom% deuterated water. The distribution of deuterated methane species is described by a simple model of the overall reaction.

We have studied the molybdenum-protein (MoFe protein) from Clostridium pasteurianum with Mössbauer spectroscopy in the temperature range from 1.5 to 200 K in magnetic fields up to 55 kG. Except for some small differences in the hyperfine parameters the results for the C. pasteurianum protein are essentially the same as those published previously for the protein from Azotobacter vinelandii, i.e. (30 ± 2) Fe atoms partition into two identical cofactor centers M (each center most likely containing six Fe atoms and one Mo atom), four P-clusters (each center containing four Fe atoms), and one iron environment labeled S (about two Fe atoms per holoenzyme). We have analyzed the spectra of the cofactor centers in three distinct oxidation states, MOX⇌e−MN→e−MR. The diamagnetic (electronic spin S = 0) state Mox is attained by oxidation of the native, EPR-active (S = 32) state MN. The reduced state MR is observed in steady state under nitrogen fixing conditions; high-field Mössbauer studies show that the cofactor centers are paramagnetic (integer electronic spin S ⩾ 1) in the state MR.

The hydrogen in methane produced by cultures of Methanobacterium thermoautotrophicum originates from water. In H2O/D2O mixtures, a methane product isotope effect is observed that increases rapidly as the water deuterium enrichment approaches 100%. This effect is due to the intracellular production of protons from H2, catalyzed by hydrogenase, which occurs at 12% the rate of water diffusion through the cell membrane. We estimate that water diffusion through the thick cell membrane of M. thermoautotrophicum is retarded by a factor of 10(6) over the free diffusion rate. The intracellular production of H+ suggests that either (1) hydrogenase is not directly involved in the production of a chemiosmotic proton gradient or (2) if it is involved, the proton gradient exists between the cytosol and the interior of vesicles observed in this bacterium. The intrinsic deutrium product isotope effect in methanogenesis is 1.20 +/- 0.1, comparable to anabolic deuterium product isotope effects in other autotrophs. An algebraic model incorporating the intracellular H2 to H+ flux accurately predicts the distribution of deuterated methane species at all levels of water deuterium enrichment.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTElectron paramagnetic resonance spectra of molybdenum(III) complexes: direct observation of molybdenum-95 hyperfine interaction and implications for molybdoenzymesB. A. Averill and W. H. Orme-JohnsonCite this: Inorg. Chem. 1980, 19, 6, 1702–1705Publication Date (Print):June 1, 1980Publication History Published online1 May 2002Published inissue 1 June 1980https://pubs.acs.org/doi/10.1021/ic50208a054https://doi.org/10.1021/ic50208a054research-articleACS PublicationsRequest reuse permissionsArticle Views602Altmetric-Citations20LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

We have investigated the role of MgATP in the reaction catalyzed by nitrogenase from Azotobacter vinelandii. There is a rapid burst of ATP hydrolysis in the pre-steady-state reaction that occurs on the same time scale as the electron transfer from dinitrogenase reductase to dinitrogenase. This burst corresponds to two ATP's hydrolyzed per electron transferred between the two proteins. Two MgATP molecules are bound to dinitrogenase reductase with dissociation constants of 430 microM and 220 microM. Investigation of the effect of MgATP concentration on the pre-steady-state kinetics of electron transfer from dinitrogenase reductase to dinitrogenase showed that there are two MgATP's required for this reaction, and the Km values are 220 microM and 970 microM. These values are similar to the dissociation constants for MgATP from dinitrogenase reductase and indicate that electron transfer between the two proteins is substantially slower than the binding and dissociation of MgATP from dinitrogenase reductase. The Km values for MgATP in steady-state H2 evolution were 390 microM and 30 microM. The decrease in the value of the second Km indicates that a slow, irreversible step occurs after the electron transfer from dinitrogenase reductase to dinitrogenase. It is possible to predict quantitatively the steady-state kinetics from the pre-steady-state kinetics, and this shows that the MgATP dependence of electron transfer is sufficient to account for effects of MgATP concentration on the steady-state H2 evolution catalyzed by nitrogenase. The hydrolysis of two ATP molecules when an electron is transferred between the two proteins of the nitrogenase system is sufficient to account for all of the ATP hydrolysis occurring in the steady-state reaction. The simplified scheme proposed to account for the MgATP dependency of the nitrogenase reaction indicates that the only role of MgATP is in support of the electron transfer from dinitrogenase reductase to dinitrogenase.