Electron‐Paramagnetic‐Resonance Studies on Nitrogenase

• Published in: European journal of biochemistry Vol. 46; no. 3; pp. 525 - 535
• Main Authors: ZUMFT, Walter G., MORTENSON, Leonard E., PALMER, Graham
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.08.1974
• DOI: 10.1111/j.1432-1033.1974.tb03646.x
• ISSN: 0014-2956

The oxidation‐reduction properties of azoferredoxin, molybdoferredoxin, and the inactive species of molybdoferredoxin, all iron‐sulfur proteins purified from Clostridium pasteurianum, were studied by potentiometry combined with electron paramagnetic resonance spectroscopy at low temperature. The technique, evaluated with spinach ferredoxin for which a midpoint potential of ‐446 mV was determined (pH 7.9, showed that azoferredoxin has a midpoint potential of ‐294 ± 20 mV (pH 7.5). In the presence of MgATP2−, azoferredoxin undergoes a negative shift of 110 mV in its oxidation‐reduction potential. The resulting potential of approximately ‐400 mV is likely to be effective under physiological conditions. The potential shift is consistent with a proposed conformational change of the protein caused by the binding of two molecules of ATP to the protein (dimer). Molybdoferredoxin and its inactive species, which has an incomplete iron‐sulfur and molybdenum centre, have midpoint potentials of approximately −20 mV and −395 mV, respectively, both at pH 7.5. All nitrogenase proteins are characterized by the transfer of one electron per redox step. Kinetic studies of the reduction of azoferredoxin and molybdoferredoxin by dithionite gave half times of <10 ms and 3–5 min, respectively. Hence, the latter process must be excluded from being part of the physiological reaction. The complex formation of ATP with azoferredoxin is a relatively slow process, which is markedly accelerated by molybdoferredoxin in the reconstituted nitrogenase system. Changes in the electron paramagnetic resonance signals of reconstituted nitrogenase were followed by a rapid‐freeze technique as a function of mixing sequence and ratio of the two proteins. The observed signal changes were independent of the mixing sequence. The steady‐state level of the molybdoferredoxin signal was also independent of the ratio of the two proteins and was less than 15% of the original resonance. The results indicate that the role of the azoferredoxin · ATP complex may not be confined solely to the function of an electron carrier for the nitrogenase reaction.