Biomedical subjects

W R Dunham

Publications and source records attributed to W R Dunham.

At least 55 records · Page 3Linked to original sources

PubMed1989

Lipoxygenase isoenzymes: a spectroscopic and structural characterization of soybean seed enzymes.

Applying recent developments in protein purification techniques, a number of lipoxygenase isoenzymes have been isolated in satisfactory quantities for a detailed physical and structural characterization. Four seed isoenzymes from two soybean cultivars have been studied by peptide mapping, free thiol and iron content determinations, and C-terminal analysis as well as by uv-visible absorption and EPR spectroscopy. While differences between the type 1 enzyme and the other isoenzymes were readily detected using proteolytic peptide mapping, digestion with dilute hydrochloric acid and C-terminal analysis both revealed structural features which were similar in all of the isoenzymes. One clear difference between the lipoxygenases was in their free sulfhydryl group content. The uv-visible absorption spectrum of each native isoenzyme was consistent with expectations for the experimental aromatic amino acid content. All of the isoenzymes contained one non-heme iron atom per molecule of protein. The oxidation of each isoenzyme with product hydroperoxide resulted in the conversion of the iron from an EPR silent state into several forms with EPR signals characteristic of high spin iron(III). The EPR spectra of all isoenzymes were remarkably similar. A time course EPR and catalytic activity study revealed that the various EPR active states represent a complex equilibrium between iron atoms in different environments. The pH dependence for the EPR and absorption spectroscopy lends support to the hypothesis that acid/base chemistry represents an important aspect of lipoxygenase catalysis.

Catalysis↗

PubMed1989

Isolated iron-molybdenum cofactor of nitrogenase exists in multiple forms in its oxidized and semi-reduced states.

Electrochemical and EPR spectroscopic experiments demonstrate that the isolated iron-molybdenum cofactor from the molybdenum-iron protein of nitrogenase from Azotobacter vinelandii exists in multiple forms in both its oxidized and semi-reduced states. The particular forms found in either oxidation state appear to be a function of the acid/base status of the solvent, N-methylformamide. In "alkaline" N-methylformamide, a single, detectable form of iron-molybdenum cofactor is observed for both oxidized and semi-reduced states. The semi-reduced form, termed R(s-r), is the one previously recognized with an S = 3/2 EPR spectrum with apparent g values of 4.6, 3.4, 2.0. Its oxidized counterpart, termed B(ox), is characterized electrochemically by a differential pulse voltammetric reduction peak at -0.37 V versus the normal hydrogen electrode. In "acidic" solvent, two distinct, previously unrecognized redox pairs of iron-molybdenum cofactor forms exist. The two semi-reduced forms, N(s-r) and W(s-r), are characterized by EPR spectra with g = 4.5, 3.6, 2.0 and g = 4.9, 3.1, 1.9, respectively. Their oxidized counterparts, A(ox) and C(ox), have differential pulse voltammetric reduction peaks at -0.32 and -0.43 V versus the normal hydrogen electrode, respectively. Manipulations of either the isolation protocol or the sample conditions affects both the type and distribution of forms present. Each form likely corresponds to a biologically significant state of the cofactor cluster within the protein.

Azotobacter↗

PubMed1989

Regional metabolism during coronary occlusion, reperfusion, and reocclusion using phosphorus31 nuclear magnetic resonance spectroscopy in the intact rabbit.

Few studies have examined metabolic consequences of coronary occlusion and reperfusion using phosphorus31 nuclear magnetic resonance (31P-NMR) in an intact animal model. Accordingly, we developed a model to study serial changes in myocardial metabolism in the intact open-chest rabbit. Ten animals underwent 20 +/- 2 minutes of regional coronary occlusion and 60 +/- 10 minutes of reperfusion followed by reocclusion. Cardiac-gated 31P-NMR spectra were obtained with a regional surface coil over the ischemic area during baseline, occlusion, reperfusion, and reocclusion conditions. Phosphocreatine fell with both the initial and second ischemic insults to 65% +/- 5% of baseline for the first occlusion (p less than 0.01) and tended to decrease to 89% +/- 8% of baseline for the second occlusion (p = 0.07), with normal levels reattained in the intervening period of reperfusion (99% +/- 5% of baseline, p = NS). Concordant inverse changes were seen with inorganic phosphates. At occlusion levels of inorganic phosphates were 135% +/- 10% of baseline (p less than 0.05) and 139% +/- 10% of baseline at reocclusion (p less than 0.05). Levels of adenosine triphosphate decreased during occlusion to 78% +/- 9% of baseline and were significantly lower than baseline during the second occlusion (75% +/- 5% of baseline, p less than 0.01). The ratio of phosphocreatine to inorganic phosphates, when compared with values at baseline, decreased at occlusion (49.6% +/- 4.7% of baseline, p less than 0.01) and at reocclusion (64.7% +/- 4.9% of baseline, p less than 0.01), with a normal ratio reattained in the intervening period of reperfusion (93.3% +/- 3.1% of baseline, p = NS). We conclude that reperfusion restores levels of phosphocreatine and adenosine triphosphate while returning levels of inorganic phosphates to baseline. Deleterious changes in high-energy phosphate metabolism are not potentiated by reocclusion in this model. 31P-NMR spectroscopy holds promise as a technique to noninvasively monitor intracellular biochemical processes serially during various interventions in the intact animal model.

Adenosine Triphosphate↗

PubMed1988

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme.

Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex. Previous work by others [Taylor, R.T., & Weissbach, H. (1969) Arch. Biochem. Biophys. 129, 745-766. Fujii, K., & Huennekens, F.M. (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum. Our enzyme preparations contain copper in approximately 1:1 stoichiometry with cobalt as determined by atomic absorption spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1988

Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex.

The exchange coupling of reduced uteroferrin has been measured (19.8(5) cm-1 S1.S2) using recently developed techniques for studying metalloprotein magnetization. A spin Hamiltonian describing the coupled binuclear Fe(II).Fe(III) center has been used to fit the low and high field magnetization data, the EPR g values, and the highly anisotropic effective hyperfine tensor of the ferric site. The exchange coupling of the phosphate complex of reduced uteroferrin has also been measured (6.0(5) cm-1 S1.S2) using the same techniques. The smaller exchange coupling of the phosphate complex is comparable with the zero field splittings of the iron sites. This results in increased sensitivity of the system g values (found by calculation from the spin Hamiltonian) to variations of the zero field splitting parameters arising from heterogeneities in the protein microenvironment. Consequently, there is a very significant (9-fold) increase in the "effective g strain" of the system compared to the situation in the absence of phosphate. This, together with the larger g anisotropy (g = (1.06, 1.51, 2.27)), gives rise to an EPR signal for the phosphate complex of reduced uteroferrin which is extremely broad and difficult to detect but which has now been identified for the first time.

Acid Phosphatase↗

PubMed1988

Modulation of progesterone synthesis and cytochrome P450 levels in rat luteal cells during human chorionic gonadotropin-induced desensitized state.

Down-regulation of hCG receptors and a steroidogenic lesion in progesterone production were observed in isolated luteal cells after 24-h treatment of pseudopregnant rats with 50 IU hCG. The luteal cells from hCG-desensitized rats exhibited a complete loss of steroidogenic response to lipoproteins in both the absence and presence of hCG. This loss of response was not overcome by the addition of 25-hydroxycholesterol. To examine if the loss of steroidogenic response is due to a transient loss of steroidogenic enzymes or inadequate delivery of substrate cholesterol, cholesterol side-chain cleavage enzyme (P450scc) activity was measured in the isolated mitochondria. No differences appeared in enzyme activity between control and hCG-treated groups. Furthermore, electron paramagnetic resonance spectra of mitochondrial cytochrome P450scc from control and hCG-treated groups exhibited no appreciable differences in low spin electron paramagnetic resonance signal (g = 1.9, 2.2, and 2.4) or high spin EPR signal (g = 8.2), suggesting that the binding of the substrate to P450scc was not impaired. Additionally, the iron sulfur protein signal (g = 1.95 and 2.03) of the reduced mitochondria remained unchanged in the desensitized group. These data were supported by immunoblotting analysis which revealed no difference in the relative amounts of iron sulfur protein or cytochrome P450scc between the two groups. Moreover, the inner mitochondrial membranes, the locus of cytochrome P450scc, showed an identical cholesterol content in control and desensitized mitochondria, indicating that the availability of cholesterol substrate is not impaired during desensitization. These results suggest that the intramitochondrial transfer of cholesterol and cholesterol side-chain cleavage enzyme activity are unaffected in the desensitized state, but an inhibitory substance might be responsible for the lack of steroidogenic response.

Animals↗

PubMed1987

Effect of aortic constriction on the functional border zone.

To evaluate how aortic constriction affects nonischemic myocardium adjacent to the perfusion boundary (the "functional border zone"), we measured systolic wall thickening (dWT) with sonomicrometers in eight anesthetized, open-chest dogs. The locations of the wall thickening measurements relative to the perfusion boundary (PB) were determined with myocardial blood flow (microspheres) maps constructed from multiple, small tissue samples. In nonischemic myocardium more than 10 mm from the PB produced by circumflex coronary occlusion, dWT increased significantly from 2.57 +/- 0.62 (mean +/- SD) to 3.24 +/- 0.73 mm (P less than 0.01). Within 10 mm of the PB, however, dWT did not change significantly (2.48 +/- 0.79 to 2.38 +/- 0.66 mm, NS). When the aorta was mechanically constricted, peak systolic pressure increased approximately 50%. Wall thickening decreased to the same relative degree in nonischemic muscle less than 10 mm and more than 10 mm from the perfusion boundary. By fitting sigmoid curves to the data, we estimated the extent of nonischemic dysfunction. It averaged 26 +/- 6 degrees (6-8 mm of endocardial circumference) during coronary occlusion alone and it was not significantly different (29 +/- 11 degrees) after aortic constriction. Thus elevated afterload affects nonischemic myocardium uniformly and does not increase the size or relative severity of the functional border zone.

Animals↗

PubMed1987

Changes in contractility fail to alter the size of the functional border zone in anesthetized dogs.

The functional border zone is nonischemic myocardium that exhibits reduced function adjacent to an ischemic area. To determine if the functional border zone can be modified by pharmacologic interventions that alter contractility, we infused isoproterenol (0.04-0.10 micrograms/kg/min) or administered propranolol (2 mg/kg) during circumflex coronary occlusion in nine anesthetized, open-chest dogs. We measured systolic wall thickening on both sides of the perfusion boundary, which was delineated with myocardial blood flow (microsphere) maps constructed from small tissue samples. By fitting sigmoid curves to the composite systolic wall thickening data after coronary occlusion, we modeled the distribution of functional impairment across the perfusion boundary. Defined as the distance from the perfusion boundary to 97.5% of the nonischemic asymptote of the sigmoid fits, the functional border zone was 31 degrees of circumference after coronary occlusion alone. Isoproterenol increased +dP/dt by 58% and augmented nonischemic systolic wall thickening without changing the lateral extent of the functional border zone (32 degrees). Propranolol reduced +dP/dt by 24% and depressed nonischemic systolic wall thickening, but the size of the functional border zone remained limited to 28 degrees. Within the functional border zone, wall thickening was significantly but only moderately reduced (-28%) compared with thickening in nonischemic myocardium more than 10 mm away from the perfusion boundary. The ratio of nonischemic border zone to central nonischemic area wall thickening remained the same with each intervention. We conclude that the dimensions of the functional border zone are fixed early after coronary occlusion and that inotropic interventions do not modify the extent or relative severity of nonischemic regional dysfunction.

Anesthesia↗

PubMed1987

Regulation of cholesterol side-chain cleavage enzyme activity by gonadotropin in rat corpus luteum.

The locus of gonadotropin-induced acute stimulation of pregnenolone production by cholesterol side-chain cleavage (CSCC) enzyme containing cytochrome P450 (cytP450scc) was examined in rat corpus luteum. Mitochondria were isolated from pseudopregnant rat ovaries after treatment with different doses of human CG (hCG) (25-200 IU) for 30 min; Electron Paramagnetic Resonance (EPR) spectra of high spin cholesterol complex of cyt P450scc (type I high spin EPR signal) and the cyt P450scc activity were determined. hCG treatment increased the formation of type I EPR spectra compared to that obtained with saline-treated controls, and pretreatment with cycloheximide (30 mg/kg BW) before hCG abolished this increase. The magnitude of type I EPR signal diminished with increasing pH over the range of 6.2-7.3. The type I EPR signal increased with doses of hCG and correlated well with the pregnenolone production. Aminoglutethimide treatment (competitive inhibitor of CSCC) before hCG injection led to an increased accumulation of cholesterol in inner mitochondrial membranes with a corresponding decrease in the outer membrane cholesterol, and cycloheximide treatment inhibited this accumulation. This suggests that the transport of cholesterol to inner mitochondrial membranes from outer membranes is regulated by hCG. In addition, gonadotropin also regulates the redistribution of cholesterol within the inner mitochondrial membranes.

Animals↗

PubMed1986

EPR of a novel high-spin component in activated hydrogenase from Desulfovibrio vulgaris (Hildenborough).

The EPR of reoxidized hydrogenase from Desulfovibrio vulgaris (H.) has been reinvestigated. In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728-3732] we find the axial signal with g = 2.06; 2.01 to be only a minor component of concentration 0.03 spin/mol. In the spectrum of fully active reoxidized enzyme this signal is overshadowed by a rhombic signal (0.1 spin/mol) with g = 2.11; 2.05; 2.00 reminiscent of the only signal found for other oxidized bidirectional hydrogenases. In addition, a novel signal has been detected with geff = 5.0 which, under the assumptions that S = 2 and [delta ms] = 2, quantitates to roughly one spin/mol. Ethylene glycol affects the relative intensity of the different signals. It is suggested that O2 sensitization parallels a spin-state transition of an iron-sulfur cluster.

Desulfovibrio↗

PubMed1986

Studies on the spin-spin interaction between flavin and iron-sulfur cluster in an iron-sulfur flavoprotein.

When the di- or trimethylamine dehydrogenases (trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7) of certain methylotrophic bacteria are reduced by two electrons with substrate unusual EPR signals arise at g = 2 and g = 4 (Steenkamp, D.J. and Beinert, H. (1982) Biochem. J. 207, 233-239; 241-252) indicative of spin-spin interaction between the FMN and iron-sulfur compounds of these enzymes. An attempt is made to understand, describe and simulate these spectra in terms of a triplet state with possible contributions from both dipolar and anisotropic exchange (J) interactions. No direct measurement of J is available, but various approaches to setting limits to J are outlined. According to these, J approximately 0.4 to 3 cm-1 or 15 to 50 cm-1. The spectra show, in the g = 2 region, a pair of rather sharp inner and a pair of broad outer lines; the latter broaden as well as move out from the center with increasing time (after substrate addition) and substrate concentration, while there is little change of g = 4. The best fits to such spectra were obtained by assuming distribution of D and E values, depending on substrate effects and arriving presumably from 'g-strain'. The fact that both shapes and intensities at g = 2 and g = 4 could be reproduced simultaneously at two frequencies indicates that the assumptions underlying our approaches and interpretations are permissible and reasonable, although we cannot claim their uniqueness. The distance between the centers of the spin densities of the flavin radical and the Fe-S cluster is thought to lie between the limits 3 to 5 A if the asymmetries in the spin-spin interaction are magnetic dipole-dipole in origin. Because there is an indication that the interaction is anisotropic exchange, the upper limit is less stringent.

Bacteria↗

PubMed1986

The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro.

A number of lipoxygenase isoenzymes were identified in developing soybean (Glycine max L. Merrill cv Provar) seeds and two have been partially characterized. In a study of lipoxygenase level in developing soybean seeds, the enzyme content increased markedly during development. Comparisons of the lipoxygenases from mature soybean seeds and immature seeds by isoelectric focusing, chromatofocusing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and peptide mapping identified two categories of isoenzyme. The isoenzymes from immature seeds were found by electron paramagnetic resonance spectroscopy to be isolated at least in part as the high spin iron(III) or active form of the enzyme in contrast to lipoxygenases from mature seeds which were isolated as electron paramagnetic resonance silent, high spin iron(II) species. The discovery of increased levels of lipoxygenases during seed development and their isolation in an active form suggests that the enzyme may play a physiological role during the maturation process. The incorporation of iron-59 from the nutrient medium into lipoxygenase during culture of immature seeds was indicative of de novo synthesis of the enzyme. The efficiency of the iron uptake was high, as indicated by the level of radioactivity found in the enzyme (one gram atom of iron per mole of lipoxygenase).

Journal Article↗

PubMed1986

The distribution of functional impairment across the lateral border of acutely ischemic myocardium.

To evaluate the degree and lateral extent of dysfunction in nonischemic myocardium adjacent to ischemic muscle, we measured systolic wall thickening with sonomicrometers during circumflex coronary occlusion in 12 anesthetized, open-chest dogs. The locations of the wall thickness measurements relative to the perfusion boundary were determined with myocardial blood flow (microspheres) maps constructed from multiple, small tissue samples. Five minutes after circumflex occlusion, systolic wall thickening in the central ischemic zone decreased from 3.00 +/- 0.61 (mean +/- SD) mm to -0.61 +/- 0.36 mm (P less than 0.01). In nonischemic myocardium greater than 10 mm from the perfusion boundary, systolic wall thickening increased from 2.56 +/- 0.57 to 3.24 +/- 0.72 mm (P less than 0.01). In nonischemic myocardium within 10 mm of the perfusion boundary, systolic wall thickening was slightly but significantly reduced compared with control (2.72 +/- 0.80 to 2.44 +/- 0.79 mm, P less than 0.05), supporting the concept of regional dysfunction in nonischemic myocardium at the lateral borders of an ischemic area. Sigmoid curves were fitted to the data to model changes in wall thickening as a continuous function of distance from the perfusion boundary. This allowed estimation of the extent of dysfunction into nonischemic myocardium which averaged less than 8 mm (approximately 30 degrees of endocardial circumference) at one border. The level of functional impairment in this zone was relatively modest, and systolic wall thickening in the immediate border area was reduced more than 50% from control only in tissue characterized by a blood supply of mixed ischemic and nonischemic origin. We conclude that a functional border zone exists lateral to an acutely ischemic area, but measurement of regional function produces relatively small exaggeration of the size of the acutely ischemic zone if severe reduction in mechanical performance is used to define the extent of the ischemic area.

Acute Disease↗

PubMed1985

A novel S = 3/2 EPR signal associated with native Fe-proteins of nitrogenase.

In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g approximately equal to 5. Temperature dependence of the signal was consistent with an S = 3/2 system with negative zero-field splitting, D = -5 +/- 0.7 cm-1. The ms = +/- 3/2 ground state doublet gives rise to a transition with geff = 5.90 and the transition within the excited ms = +/- 1/2 doublet has a split geff = 4.8, 3.4. Quantitation gave 0.6 to 0.8 spin . mol-1 which summed with the spin intensity of the S = 1/2 g = 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S = 3/2 signal with no concomitant changes in intensity of the S = 1/2 signal.

Adenosine Triphosphate↗

PubMed1985

Spectroscopic studies on the iron-sulfur centers of milk xanthine oxidase.

The optical electron paramagnetic resonance and Mössbauer spectral properties of the two iron-sulfur centers present in milk xanthine oxidase have been reexamined. It is found in the case of the optical spectral change observed on reduction of the enzyme that the two centers contribute approximately equally, with a ratio of spectral contributions for Fe/S I and Fe/S II of 0.55:0.45. This conclusion is based both on the behavior of the spectral change at wavelengths where only the two iron-sulfur centers contribute to the spectral change (under experimental conditions minimizing the effect of flavin semiquinone) during reductive titrations and a comparison of the spectra of 1- and 2-electron reduced enzyme under different conditions. This very similar spectral weighting for the two centers applies throughout the visible region. In the case of the EPR spectra, it is found from computer simulation of the signals observed under nonsaturating conditions that iron-sulfur center II exhibits g values of 1.902, 1.991, and 2.110 and does not exhibit two g values above that for the free electron, as has been reported (Lowe, J., Lynden-Bell, R.M., and Bray, R. C. (1972) Biochem. J. 130, 239-249). The g values for iron-sulfur center I obtained from the simulations are 1.894, 1.932, and 2.022. Finally, Mössbauer spectra of xanthine oxidase have been obtained, and it is found that while the two iron-sulfur centers are indistinguishable in the oxidized state, the ferrous iron in one of the reduced iron-sulfur centers exhibits an unusually large quadrupole coupling.

Animals↗

PubMed1985

On the prosthetic group(s) of component II from nitrogenase. EPR of the Fe-protein from Azotobacter vinelandii.

The EPR spectrum of the reduced Fe-protein from nitrogenase has been reinvestigated. The dependences on temperature, microwave power, and microwave frequency all suggest that the observed signal represents a magnetically isolated [4Fe-4S]1+(2+;1+) cluster. Also, the signal can be simulated assuming a simple, g-strained S = 1/2 system. However, the integrated intensity amounts to no more than 0.2 spins per protein molecule. It is, therefore, impossible that Fe-protein preparations contain a single type of [4Fe-4S] cluster.

Azotobacter↗

PubMed1985

Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin. Possible new fingerprints for three iron clusters.

We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus. First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers. This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2. Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the [4Fe-4S]+ (S = 1/2) and the reduced 3Fe center. A theoretical explanation is given for the observation of interaction signals with constant effective g values.

Electron Spin Resonance Spectroscopy↗

PubMed1985

The importance of quantitative Mössbauer spectroscopy of MoFe-protein from Azotobacter vinelandii.

The Mössbauer spectra of MoFe-protein of Azotobacter vinelandii, as isolated under dithionite and taken at temperatures from 125 K to 175 K, are the sums of four resolved quadrupole doublets. Our results indicate that the currently accepted interpretation of these doublets can be questioned. Our data reduction method converts the Mössbauer transmission spectra to source lineshape deconvolved absorption spectra linear in iron. We used these absorption spectra to determine the stoichiometry of the Fe clusters in MoFe-protein and we obtained much better fits if we assumed that there are four iron atoms in the 'Fe2+, doublet, two iron atoms in the 'S' doublet, twelve iron atoms in the 'D' doublet and sixteen iron atoms in the 'M' doublet. Therefore we propose that the MoFe-cofactor contains one molybdenum and eight iron atoms ('M'). We also argue that none of the previous Mössbauer spectroscopic studies have been performed on the highest-activity preparation now obtainable, nor has there been any study to prove that the Mössbauer spectra are independent of activity. We consider that the Mössbauer spectroscopic studies of the MoFe-protein of nitrogenase are a re-opened and unsolved problem.

Azotobacter↗