Third-harmonic detection of electron-paramagnetic-resonance spectra: Resolution of the hyperfine splitting in nitrosylated nitrite reductase from vegetable marrow (Cucurbita pepo)

1980 ◽  
Vol 8 (5) ◽  
pp. 642-642 ◽  
Author(s):  
RICHARD CAMMACK ◽  
IAN V. FRY
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Nitrite Reductase ◽  
Hyperfine Splitting ◽  
Cucurbita Pepo ◽  
Harmonic Detection ◽  
Paramagnetic Resonance ◽  
Third Harmonic ◽  
Vegetable Marrow ◽  
Electron Paramagnetic

scholarly journals Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron–sulphur centre and haem under turnover conditions

1978 ◽  
Vol 171 (3) ◽  
pp. 519-526 ◽  
Author(s):  
Richard Cammack ◽  
Dereck P. Hucklesby ◽  
Eric J. Hewitt
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Low Temperature ◽  
Nitrite Reductase ◽  
Cucurbita Pepo ◽  
Methyl Viologen ◽  
Reaction Sequence ◽  
Paramagnetic Resonance ◽  
Midpoint Potential ◽  
Vegetable Marrow ◽  
Electron Paramagnetic

Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe3+ in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be −120mV. On reduction with Na2S2O4 or Na2S2O4+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron–sulphur centre. The midpoint potential of this centre was very low, about −570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na2S2O4, the iron–sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron–sulphur centre. Other ligands to haem, such as CO and the reaction product NH3, had similar but less pronounced effects, and also changed the lineshape of the iron–sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na2S2O4 in various proportions. Signals were interpreted as due to the reduced iron–sulphur centre (with slightly different g values), a haem–NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem–NO spectrum was more intense, whereas in the presence of an excess of Na2S2O4 it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem–NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH4+.

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

1986 ◽  
Vol 64 (5) ◽  
pp. 394-399 ◽  
Author(s):  
Jai C. Sadana ◽  
Bashir M. Khan ◽  
Ian V. Fry ◽  
Richard Cammack
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance ◽  
Nitrite Reductase ◽  
Hyperfine Splitting ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Compound A ◽  
Electron Paramagnetic ◽  
Nitrosyl Derivative

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.

Electron paramagnetic resonance and INDO study of carbon-13 hyperfine splitting constants in benzyl radical and toluene radical anion

1975 ◽  
Vol 97 (16) ◽  
pp. 4477-4482 ◽  
Author(s):  
Arthur M. Ihrig ◽  
Paul Ronald Jones ◽  
Il Nam Jung ◽  
Roger V. Lloyd ◽  
James L. Marshall ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Hyperfine Splitting ◽  
Radical Anion ◽  
Paramagnetic Resonance ◽  
Benzyl Radical ◽  
Electron Paramagnetic

scholarly journals Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins

1980 ◽  
Vol 189 (2) ◽  
pp. 285-294 ◽  
Author(s):  
M K Johnson ◽  
A J Thomson ◽  
T A Walsh ◽  
D Barber ◽  
C Greenwood
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Absorption Spectra ◽  
High Temperatures ◽  
Nitrite Reductase ◽  
Axial Ligand ◽  
Coupling Constants ◽  
Paramagnetic Resonance ◽  
Temperature Spectra ◽  
Hyperfine Splittings ◽  
Electron Paramagnetic

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.

Sign in / Sign up

Export Citation Format

Share Document