Properties of Terminal Oxidase Systems of Bacteroids from Root Nodules of Soybean and Cowpea and of N2-fixing Bacteria Grown in Continuous Culture

Abstract

235The respiration of bacteroids from root nodules of soybean (inoculated with Rhizobium japonicum strain CBlSO9) and cowpea (inoculated with Rhizobium sp. strain CB756) was studied at low concentrations of dissolved 0, in a stirred electrode chamber (range 0.1 to 10 ,uM-OJ and during deoxygenation of solutions containing oxyleghaemoglobin (range 0.003 to 0-3 PM-O,) or oxymyoglobin (0.1 to 10 pM-0,). The 0, affinity of terminal oxidase systems of these bacteria (measured as apparent Ks) depended on the range of concentration of free, dissolved 0, in which the measurements were made. This was due to the capacity of the oxidases to express increasing affinity as the 0, concentration declined below certain transition concentrations, thus tending to maintain the respiration rate. In the lowest concentration range (0-003 to 0.01 p M -0 , ) an oxidase of very high affinity for 0, (apparent Ks 0.005 ,UM) appeared to be under allosteric control. Three additional oxidases or oxidase affinity states could be recognized. In strain CB1809, the one with lowest affinity was insensitive to CO and less sensitive to N-phenylimidazole and azide, whilst the oxidases of higher affinity were very sensitive to these inhibitors. Terminal oxidase systems of the same strains from 0,-limited continuous cultures resembled those of bacteroids, when assayed in the same way.The properties of terminal oxidases of Azotobacter vinelandii strain AVO, Azospirillum brasilense strain Sp7 and Klebsiella pneumoniae strain 5023 1, when grown in 0,-limited continuous cultures, were examined in the same experimental systems. In all culture states examined there was no evidence for multiple oxidases as seen with the Rhizobium spp. In two of these bacteria, Azospirillum brasilense and K. pneumoniae, the oxidases appeared to be allosteric but their affinities were very different (apparent Ks 0.006 and 0.1 1 ,UM, respectively). The terminal oxidase of Azotobacter vinelandii obeyed Michaelis-Menten kinetics, but had a lower affinity for O2 (apparent Ks 0.48 ,LAM).

Determination of the oxygen affinities of terminal oxidases in Azotobacter vinelandii using the deoxygenation of oxyleghaemoglobin and oxymyoglobin: cytochrome bd is a low-affinity oxidase

“…The values of K' (= k, oxygen dissociation rate/k', association rate) used in these calculations were 786 x lo-' M (oxymyoglobin) or 50 x lo-' M (oxyleghaemoglobin) (Appleby & Bergersen, 1980). Plots of 1 / I./ versus 1 /S (Lineweaver-Burke) and of I'versus V/S (Eadie-Hofstee) were used to calculate K , values.…”

“…The concentration of myoglobin was determined by CO difference spectroscopy at room temperature using a Johnson Foundation SDB3 dual-wavelength scanning spectrophotometer and an absorption coefficient of 177 mM-' cm-' at 435-420 nm (Wood, 1984). The concentration of leghaemoglobin was determined from the absolute spectrum of the oxygenated form using the same instrument and an absorption coefficient of 14 mM-l cm-' (575-700 nm) (Appleby & Bergersen, 1980). Samples of globins were diluted to 10--20 pM in potassium phosphate buffer (25 mM, pH 7.0) containing 1 mM EDTA, which had been previously sparged with a gas mixture (BOC) of 1 % oxygen in argon.…”

“…Oxygenated soybean leghaemoglobin (a kind gift from Dr F. Bergersen, Division of Plant Industry, CSIRO, Canberra, Australia) and the oxygenated form of sperm whale myoglobin (Sigma, no longer available) were prepared using published methods (Appleby & Bergersen, 1980). The concentration of myoglobin was determined by CO difference spectroscopy at room temperature using a Johnson Foundation SDB3 dual-wavelength scanning spectrophotometer and an absorption coefficient of 177 mM-' cm-' at 435-420 nm (Wood, 1984).…”

Effect of Carotenoid Overproduction on Oxygen Tolerance of Nitrogen Fixation in Azospirillum brasilense Sp7

“…Azospirillm brasilense has rather high respiratory activity, and it adapts to different oxygen concentrations (Bergersen & Turner, 1980;Okon et al, 1983) and carries enzymes that detoxify superoxide and hydrogen peroxide Moore et al, 1984). However, unlike Azotobacter spp., AzospirilIurn does not possess an efficient respiratory and conformational protection of nitrogenase (Robson & Postgate, 1980;Yates & Jones, 1974;Nelson & Knowles, 1978;Hochman et al, 1987).…”

Effects of Carbohydrate on the Internal Oxygen Concentration, Oxygen Uptake, and Nitrogenase Activity in Detached Pea Nodules

“…F=(0e-O)/R (1) 'Supported by Department of Energy contract 82ER-12066 to T. A. L. where the flux density (flux per unit area) (F) of 02 across the diffusion barrier is proportional to the difference between the concentration outside (Qe) and inside (0,) the nodule, and inversely proportional to the resistance (R) to 02 diffusion in the cortex (9).…”