scholarly journals Electron-paramagnetic-resonance and magnetic-circular-dichroism studies on the formate dehydrogenase-nitrate reductase particle from Pseudomonas aeruginosa

1987 ◽  
Vol 243 (1) ◽  
pp. 241-248 ◽  
Author(s):  
C Godfrey ◽  
P M A Gadsby ◽  
A J Thomson ◽  
C Greenwood ◽  
A Coddington
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Transfer ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Magnetic Circular Dichroism ◽  
Paramagnetic Resonance ◽  
Membrane Bound ◽  
Electron Paramagnetic ◽  
Transfer Chain ◽  
Mediate Electron Transfer

The membrane-bound respiratory particle complex of Pseudomonas aeruginosa, which reduces nitrate to nitrite using formate as the electron donor, was prepared and characterized by e.p.r. and low-temperature magnetic c.d. (m.c.d.) spectroscopy. The particle complex has two enzymic components, namely nitrate reductase (NiR) and formate dehydrogenase (FDH), which are multi-centred proteins containing molybdenum, iron-sulphur clusters and cytochrome. By using results from work on the purified extracted enzymes NiR and FDH to aid in the assignment, it has been possible to observe spectroscopically all the components of the electron-transfer chain in the intact particle. This led to a proposal for the organization of the metal components of the FDH-NiR chain. Molybdenum ions are at opposite ends of the chain and interact with, respectively, the formate-CO2 couple and the nitrate-nitrite couple. The molybdenum ion at the low-potential end of the chain passes electrons to cytochrome b of FDH, a bishistidine-co-ordinated haem with unusual steric restraint at the iron. The next component is a [4Fe-4S] cluster. This comprises all the components of FDH. Electrons are passed to the molybdenum of NiR via a number, probably two, of [4Fe-4S] clusters. No evidence has been found in this work for the presence of a quinone to mediate electron transfer between FDH and NiR. Cytochrome c appears to be able to feed electrons into the chain at the level of one of the [4Fe-4S] centres of NiR.

scholarly journals Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre

1987 ◽  
Vol 243 (1) ◽  
pp. 235-239 ◽  
Author(s):  
P M A Gadsby ◽  
C Greenwood ◽  
A Coddington ◽  
A J Thomson ◽  
C Godfrey
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Electron Paramagnetic Resonance ◽  
Hyperfine Coupling ◽  
Formate Dehydrogenase ◽  
Sodium Dithionite ◽  
Paramagnetic Resonance ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Electron Paramagnetic ◽  
Fold Excess

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.

scholarly journals Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Characterization of haem and iron-sulphur centres by magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy

1987 ◽  
Vol 243 (1) ◽  
pp. 225-233 ◽  
Author(s):  
C Godfrey ◽  
A Coddington ◽  
C Greenwood ◽  
A J Thomson ◽  
P M A Gadsby
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Low Temperature ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Magnetic Circular Dichroism ◽  
Sodium Dithionite ◽  
Anaerobic Growth ◽  
Organic Radical ◽  
Resonance Spectroscopy ◽  
Reduced State

The purification of formate dehydrogenase (FDH) from Pseudomonas aeruginosa after anaerobic growth on nitrate-containing medium was carried out. The separation of the FDH enzyme from nitrate reductase (NiR), which are found together in a particle fraction and constitute the short respiratory chain of this bacterium, has been followed by optical, magnetic c.d. (m.c.d.) and e.p.r. spectroscopy. These techniques have allowed the haem, iron-sulphur clusters and molybdenum components to be detected and, in part, their nature to be determined. Attempts to extract FDH anaerobically in the absence of sodium dithionite led to loss of activity. Addition of sodium dithionite maintained the activity of the enzyme, even after subsequent exposure to air, in an assay involving formate reduction with Nitro Blue Tetrazolium as reductant. Three preparations of FDH have been examined spectroscopically. The preparations vary in the amount of contaminating nitrate reductase, the amount of cytochrome c present and the concentration of oxidized [3Fe-4S] cluster. Optical spectra and low-temperature m.c.d. spectroscopy show the loss of a cytochrome-containing protohaem IX co-ordinated by methionine and histidine as NiR is separated from the preparation. In its purest state FDH contains one molecule of cytochrome co-ordinated by two histidine ligands in the oxidized state. This cytochrome has an e.p.r. spectrum with gz = 3.77, the band having the unusual ramp shape characteristic of highly anisotropic low-spin ferric haem. It also shows a charge-transfer band of high intensity in the m.c.d. spectrum at 1545 nm. It has recently been shown [Gadsby & Thomson (1986) FEBS Lett. 197, 253-257] that these spectroscopic properties are diagnostic of a bishistidine co-ordinated haem with steric constraint of the axial ligands. The e.p.r. and m.c.d. spectra of the reduced state of FDH reveal the presence of an iron-sulphur cluster of the [4Fe-4S]+ type. The g-values are 2.044, 1.943 and 1.903. An iron-sulphur cluster of the class [3Fe-4S], detected by e.p.r. spectroscopy in the oxidized state and by low-temperature m.c.d. spectroscopy in the reduced state, is purified away with the NiR. Finally, an e.p.r. signal at g = 2.0 with a narrow bandwidth which persists to 80 K is observed in the purest preparation of FDH. This may arise from an organic radical species.

Sign in / Sign up

Export Citation Format

Share Document