scholarly journals Spectroscopic, thermodynamic and kinetic properties of Candida nitratophila nitrate reductase

1990 ◽  
Vol 272 (2) ◽  
pp. 545-548 ◽  
Author(s):  
C J Kay ◽  
M J Barber ◽  
L P Solomonson ◽  
D Kau ◽  
A C Cannons ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Nitrate Reductase ◽  
Chlorella Vulgaris ◽  
Close Similarity ◽  
Kinetic Properties ◽  
Potentiometric Titrations ◽  
Oxidation Reduction ◽  
Visible Spectra ◽  
Rate Limiting ◽  
Prosthetic Groups

Visible spectra of oxidized and reduced Candida nitratophila assimilatory NAD(P)H:nitrate reductase yielded absorbance maxima of 413 nm and 423 nm, and 525 nm and 555 nm respectively, characteristic of a b5-type cytochrome. E.p.r. spectra of the partially reduced enzyme revealed a single Mo(V) species (g1 = 1.9957, g2 = 1.9664 and g3 = 1.9658) exhibiting superhyperfine coupling to a single proton [A(1H)av. = 1.4 mT]. Oxidation-reduction midpoint potentials (E'0) (25 degrees C, pH 7) for the haem and Mo-pterin prosthetic groups were determined by visible and e.p.r. potentiometric titrations and yielded values of E'0 = -174 mV (n = 1) for the haem and E'0 = -3 mV and E'0 = -27 mV for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples respectively. Comparison of initial rates of the NADH-oxidizing and nitrate-reducing partial activities at various ionic strengths indicated electron transfer from reduced haem to Mo was rate-limiting during turnover. These results suggest a close similarity between Candida nitratophila and Chlorella vulgaris nitrate reductases.

scholarly journals Oxidation–reduction midpoint potentials of the flavin, haem and Mo-pterin centres in spinach (Spinacia oleracea L.) nitrate reductase

1989 ◽  
Vol 263 (1) ◽  
pp. 285-287 ◽  
Author(s):  
C J Kay ◽  
M J Barber ◽  
B A Notton ◽  
L P Solomonson
Keyword(s):  
Nitrate Reductase ◽  
Room Temperature ◽  
Spinacia Oleracea ◽  
Midpoint Potential ◽  
Redox Couples ◽  
Oxidation Reduction ◽  
Spinacia Oleracea L ◽  
Unicellular Green Alga ◽  
Green Alga Chlorella ◽  
Prosthetic Groups

Oxidation-reduction midpoint potentials have been determined for the flavin, cytochrome b557 and Mo-pterin prosthetic groups of spinach (Spinacia oleracea L.) assimilatory nitrate reductase using visible, c.d. and room-temperature e.p.r. potentiometric titrations. At pH 7 and 25 degrees C, the midpoint potential for the FAD/FADH2 couple was determined by c.d. potentiometry to be -280 +/- 10 mV (n = 2). The redox potential for reduction of the haem was determined by visible potentiometry to be -123 +/- 10 mV (n = 1), significantly lower than the previously published value of -60 mV [Fido, Hewitt, Notton, Jones & Nasrulhaq-Boyce (1979) FEBS Lett. 99, 180-182]. Potentials for the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) redox couples, determined by room-temperature e.p.r. potentiometry, were found to be +2 +/- 20 and -6 +/- 20 mV respectively. These values are very similar to the values previously determined for the FAD, haem and Mo-pterin centres in assimilatory nitrate reductase isolated from the unicellular green alga Chlorella vulgaris and indicate a close thermodynamic similarity between the two enzymes.

scholarly journals Measurement of the oxidation-reduction potentials for one-electron and two-electron reduction of electron-transfer flavoprotein from pig liver

1984 ◽  
Vol 219 (3) ◽  
pp. 1043-1047 ◽  
Author(s):  
M Husain ◽  
M T Stankovich ◽  
B G Fox
Keyword(s):  
Electron Transfer ◽  
Pig Liver ◽  
Electron Transfer Flavoprotein ◽  
Midpoint Potential ◽  
Potentiometric Titrations ◽  
Reduction Potentials ◽  
Oxidation Reduction ◽  
Electron Reduction ◽  
Reducing Equivalents

Potentiometric titrations of pig liver electron-transfer flavoprotein (ETF) were performed at pH 7.5 and 4 degrees C, both in the reductive and oxidative directions. Reduction of ETF to the hydroquinone form required a total of two reducing equivalents/mol of ETF with the formation of sub-stoichiometric amounts of anionic semiquinone as an intermediate. The oxidation-reduction potentials for the two one-electron couples, oxidized ETF/ETF semiquinone and ETF semiquinone/fully reduced ETF, are +4 mV and -50 mV respectively. The overall midpoint potential for the two-electron couple (oxidized ETF/fully reduced ETF) is -23 mV.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals The Presence of Bound Cyanide in the Naturally Inactivated Form of Nitrate Reductase of Chlorella vulgaris

1974 ◽  
Vol 249 (19) ◽  
pp. 6074-6079 ◽  
Author(s):  
George H. Lorimer ◽  
Hans-Siegfried Gewitz ◽  
Wolfgang Völker ◽  
Larry P. Solomonson ◽  
Birgit Vennesland
Keyword(s):  
Nitrate Reductase ◽  
Chlorella Vulgaris

scholarly journals A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath)

1989 ◽  
Vol 259 (1) ◽  
pp. 167-172 ◽  
Author(s):  
J Green ◽  
H Dalton
Keyword(s):  
Electron Transfer ◽  
Protein C ◽  
Turnover Rate ◽  
Protein A ◽  
Methylococcus Capsulatus ◽  
Bond Breakage ◽  
Conversion Of Methane ◽  
Protein Components ◽  
A Charge ◽  
Rate Limiting

1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol.

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