Electron-paramagnetic-resonance studies of leghaemoglobins from soya-bean and cowpea root nodules. Identification of nitrosyl-leghaemoglobin in crude leghaemoglobin preparations

Abstract

1. Leghaemoglobins from soya-bean (Glycine max) and cowpea (Vigna unguiculata) root nodules were purified by chromatography on DEAE-cellulose phosphate columns at pH8.0 and pH5.8, to avoid the relatively low pH (5.2) commonly used to purify these proteins. 2. E.p.r. (electron-paramagnetic-resonance) spectra of the fluoride, azide, hydroxide and cyanide complexes of these ferric leghaemoglobins were very similar to the spectra of the corresponding myoglobin derivatives, indicating that the immediate environment of the iron in leghaemoglobin and myoglobin is similar, an imidazole moiety of histidine being the proximal ligand to the haem iron [cf. Appleby, Blumberg, Peisach, Wittenberg & Wittenberg (1976) J. Biol. Chem.251, 6090-6096]. 3. E.p.r. spectra of the acid-metleghaemoglobins showed prominent high-spin features very near g=6 and g=2 and, unlike myoglobin, small low-spin absorptions near g=2.26, 2.72 and 3.14. The width of the g=6 absorption derivative at 10-20K was about 4-4.5mT, similar to the value for acid-methaemoglobin. In contrast, a recently published (Appleby et al., 1976) spectrum of acid-metleghaemoglobin a had less high-spin character and a much broader absorption derivative around g=6. 4. E.p.r. spectra of ferric leghaemoglobin nicotinate and imidazole complexes suggest that the low-spin absorption near g=3.14 can be attributed to a trace of ferric leghaemoglobin nicotinate, and those near g=2.26 and 2.72 are from an endogenous dihistidyl haemichrome. 5. A large e.p.r. signal at g=2 in all samples of crude leghaemoglobin was shown to be from nitrosyl-leghaemoglobin. A soya-bean sample contained 27+/-3% of the latter. A previously unidentified form of soya-bean ferrous leghaemoglobin a was shown to be its nitrosyl derivative. If this is not an artifact, and occurs in the root nodule, the nitrosyl radical may interfere with the function of leghaemoglobin.

Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

“…This complex was unequivocally identified on the basis of its c~, 13 and 7 peaks at 570, 544 and 413.5 nm (Fig. 3), in agreement with the values reported for the Lb 2 + 9 NO complex generated from Lb 3 +, nitrite and Na/S204 (Dilworth and Appleby 1974;Maskall et al 1977;Kanayama and Yamamoto 1990).…”

“…In anaerobic conditions the system NADH + Rfl reduced nitrite to NO, as shown by the formation of Lb 2 + -NO (Table 4). This complex has been detected by electron-paramagnetic-resonance spectroscopy (Maskall et al 1977) or spectrophotometry (Kanayama and Yamamoto 1990) of nodule extracts from plants fed with nitrate. Recently, Kanayama and Yamamoto (1990) have put forward the hypothesis that the accumulation of Lb 1+.…”

Enzymatic and nonenzymatic mechanisms for ferric leghemoglobin reduction in legume root nodules.

“…Addition of high amounts of SOD (50 ,ug) had little effect on the rate of Lb3+ reduction, but catalase (6 ,ug) inhibited FLbR activity by 80% throughout the incubation period (Table 1) ,(, and y absorption bands at 558-560, 537.5-538.5, and 410-410.5 nm, respectively, for Lbs from different species. These spectral characteristics obviously differ from those of Lb2+-NO (7,21). To our knowledge, this is the first time a complex between Lb3+ and NO2-has been described, but a similar complex has been reported for Hb3+ (22).…”

Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus

“…Nitric oxide is an inhibitor of nitrogenase from nitrogen-fixing bacteria, through interaction with Fe-S clusters [24]. NO binds tightly to leghemoglobin, forming nitrosylleghemoglobin complexes [25]. It has been reported that accumulation of nitrosylleghemoglobin in nodules of leguminous plants supplied with nitrate inhibits nitrogenase activity [26].…”