Leonard E. Mortenson

Purification and properties of hydrogenase from Clostridium pasteurianum W5.

Hydrogenase from Clostridium pasteurianum W5 prepared by a previously reported method was found to be heterogeneous. A new procedure has been developed which achieved a 320-fold purification with an 11% recovery of the enzyme activity. This represents a 6.6-fold increase in specific activity over the previously reported value. Such preparations showed a maximal activity (V) of about 4000 μmoles H2 evolved/min/mg protein when assayed in the presence of ferredoxin and 1 mM methyl viologen. Hydrogenase so prepared has a molecular weight of 60 500 and did not dissociate into subunits when treated with sodium dodecylsulfate and 2-mercaptoethanol. Hydrogenase has 12 half-cystine residues per molecule and a preponderance of acidic amino acids. This hydrogenase does not contain molybdenum or copper but has about 12 iron atoms and 12 acid-labile sulfur groups per molecule. All of the iron atoms are resistant to anaerobic treatment with o-phenanthroline. Methyl viologen, benzyl viologen, methylene blue and ferredoxin can each serve as the sole electron carrier for the purified enzyme.

Purification and analysis of ferredoxin from Clostridium pasteurianum

Abstract Ferredoxin from Clostridium pasteurianum has been obtained in a pure state by a new rapid method employing the fact that Fd is soluble in 50–60% acetone while over 99% of the material in dry cells is not. Crystalline Fd purified 918-fold over the dry cells can be obtained in 3 days or less with this method. The amino acid composition and iron and sulfide content of Fd purified by this method are presented.

Inhibition of methylene blue formation during determination of the acid-labile sulfide of iron-sulfur protein samples containing dithionite

Abstract The acid-labile sulfide content of iron-sulfur proteins is commonly determined colorimetrically by reacting the released sulfide with N,N′ -dimethyl- p -phenylenediamine and FeCl 3 , under acidic conditions, to form methylene blue. This reaction was found to be inhibited strongly when the iron-sulfur protein samples contained dithionite, which was frequently added during the purification of these proteins to prevent oxygen damage. The inhibitory substance(s) seems to be an anaerobic decomposition product(s) of dithionite. The use of concentrated protein solutions and small sample volumes was found to be the most effective way of overcoming this problem. This simple principle should also be useful when analyzing crude samples which may contain other low molecular weight, interfering substances.

Electron paramagnetic resonance studies on nitrogenase. II. Interaction of adenosine 5′-triphosphate with azoferredoxin

The interaction of ATP with both iron-sulfur proteins of nitrogenase from Clostridium pasteurianum, azoferredoxin and molybdoferredoxin, has been studied by low-temperature EPR spectroscopy. ATP in the presence of Mg2+ changes the rhombic EPR signal of azoferredoxin with g-values of 2.06, 1.94 and 1.87 to an axial signal, with g values of 2.04 and 1.93. The binding of two molecules of ATP per azoferredoxin dimer (mol. wt 55 000) is suggested. Comparative data with other purine and pyrimidine nucleotides and ATP analogues demonstrate the involvement of structural elements of the substrate in the conversion of the EPR signal of azoferredoxin. A similar effect is induced by 5 M urea, which suggests that ATP causes a conformation change of the protein. In contrast, no effect of ATP was observed on the EPR signal of molybdoferredoxin.

Purification, metal composition and properties of molybdo-ferredoxin and azoferredoxin, two of the components of the nitrogen-fixing system of Clostridium pasteurianum☆

Abstract A procedure for the separation and purification of two components of Clostridium pasteurianum involved in N2 fixation are presented. One of the components, molybdo-ferrodoxin, was about 78% pure based on a molybdenum content of 1 atom per molecule and a mol. wt. of 100 000. At this stage of purity it contained per Mo atom 1 atom Mg, 12 atoms of Fe and 3 atoms of sulfide. The second component, azoferredoxin was purified 30–50-fold and contained Fe and sulfide. Both components were inactivated by O2. Molybdoferredoxin could be frozen anaerobically with little loss in activity whereas in contrast azoferredoxin lost partial activity under all conditions of storage examined. Neither molybdoferredoxin nor azoferredoxin was present in extracts of cells grown on NH3.

Purification and properties of hydrogenase, an iron sulfur protein, from Clostridium pasteurianum W5

Abstract 1. 1. Hydrogenase (H2:ferredoxin oxidoreductase, EC 1.12.1.1) from Clostridium pasteurianum W5 has been purified to a state that shows one protein band with one coincident activity peak on disc electrophoresis on polyacrylamide gel and a single peak in the analytical ultracentrifuge. 2. 2. The purified enzyme has a molecular weight of 60 000 and an isoelectric point of 5.0, and it contains 4.0 iron and 4.0 acid-labile sulfide groups per molecule.

Components of cell-free extracts of clostridium pasteurianum required for ATP-dependent H2 evolution from dithionite and for N2 fixation

Abstract The same two protein components are required for ATP-dependent H 2 evolution as were shown previously to be required for N 2 fixation and electron-dependent ATP utilization. One of those components contains molybdenum, iron, and inorganic sulfide while the other contains iron and sulfide. Quantitative data indicated that ATP may be required for electron transport from one component to the other. A third component may also be required. A proposed mechanism for N 2 fixation based on present knowledge is presented.

Ferredoxin requirement for nitrogen fixation by extracts of clostridium pasteurianum

Abstract Ferrodoxin has been found to be an absolute requirement for N 2 -fixation in extracts of Clostridium pasteurianum with pyruvate as the electron donor. In addition to ferredoxin other requirements for N 2 -fixation have been demonstrated but not identified.

Determination of molybdenum and tungsten in biological materials

Abstract A spectrophotometric method for the quantitative determination of molybdenum and tungsten in the presence of each other in biological materials, has been developed. The method involves the selective formation and extraction of the complexes of the metals with 4-methyl-1,2-dimercaptobenzene under specified conditions of acidity and temperature. Possible interference by components found in biological materials, was examined and shown to be negligible.

Acetylene reduction by nitrogen fixing extracts of Clostridium pasteurianum: ATP requirement and inhibition by ADP.

NITROGEN fixing extracts of Clostridium pasteurianum have been shown to catalyse the reduction of acetylene1,2. The requirements for this reaction were shown to be the same as those for nitrogen fixation, that is, a low potential electron source, ATP, an ATP generating system and magnesium ions. Acetylene reduction provides a more sensitive assay for the activity of the nitrogen fixing system than ammonium production and the measurement of rates of reactions is facilitated. The results described in this communication show that the reduction of acetylene by these extracts can be supported directly by ATP without an ATP generating system. When an ATP generating system is not used we now find that (a) magnesium ions are required for ATP utilization by the nitrogen fixing system, a fact not previously demonstrated, for magnesium ions were required for the ATP-generating system; that (b) the acetylene reduction by the nitrogen fixing system is inhibited by ADP in a manner which suggests that ADP is a negative modifier; and that (c) the acetylene reducing system needs to interact with more than one molecule of ATP.