The Respiratory System of Azotobacter vinelandii

Abstract

1. A detailed procedure is described for the isolation of stable respiratory membranes of relatively high phosphorylation efficiency from nitrogen-fixing cultures of Azotobacter vinelandii 2 . Three phosphorylation sites are detectable. These are located a t the level of NADH dehydrogenase (Site I; P/2e 0.45)) between the primary dehydrogenases and the cytochromes (Site 11; P/2e 0.53) and on the minor terminal pathway, to the oxygen side of the c-type cytochromes (Site 111; P/2e 0.22).3. Switching of electron flow between the terminal oxidation pathways with low concentrations of KCN and 2-n-alkyl-4-hydroxyquinoline-N-oxide causes changes in oxygen uptake and phosphate esterification which are compatible with the postulated locations of the three phosphorylation sites and which demonstrate, indirectly, the non-phosphorylating nature of the b, --f a2 major terminal pathway.4. Phosphorylation efficiences with ascorbate plus purified A . vinehndii cytochromes c4 and c5, horse heart cytochrome c and the artificial electron mediators 2,6-dichlorophenol indophenol and N,N,N',N'-tetramethyl-p-phenylene diamine indicate that the minor terminal pathway is further split into a slow, phosphorylating pathway (c5 --f oxygen) and a faster, non-phosphorylating pathway (c4 -+ oxygen).(P/O, NADH = 0.90-1.10).The efficiencies of oxidative phosphorylation exhibited by isolated bacterial respiratory membranes are in general very much lower than those of intact mammalian mitochondria [l].Cell free extracts of gram positive bacteria (e. g. Mycobacterium phlei [2], Corynebacterium creatovorans[3] and Micrococcus lysodeikticzcs [4]) exhibit high P/O ratios (up to 1.8 when oxidising succinate or malate, but lower with NADH) ; isolated respiratory membranes are less efficient, but can be stimulated to yield higher P/O ratios by the addition of soluble factors [2,4,5]. Of the gram negative bacteria so far studied, the chemoautotrophs (e. g . Nitrobacter agilis [6], Nitrobacter winogradskii [7]) yield respiratory membranes with highly efficient, self contained oxidative phosphorylation systems (P/O ratios up to 2.8 with NADH), whereas the heterotrophs (e. g . Escherichia coli [8,9]. Alcaligenes fuecalis [lo] and Azotobacter vinelandii [ l l , 121) in general yield respiratory membranes of much lower phosphorylation efficiency (P/O ratios with NADH rarely exceed 0.5), even when optimally supplemented with soluble factors.Definition. A,,, unit, the quantity af material contained in i m l of a solution which has an absorbance of i a t 680 nm when measured in a i-cm path length cell.The membrane-bound respiratory system of Azotobucter vinehndii is characterised by extremely high oxidase activities, particularly with NADH or malate, a rich and varied content of electron carriers and a branched terminal cytochrome system [13-161. The nature of this branched cytochrome system was elucidated via kinetic and inhibitor studies which suggested that the major electron flow was catalysed by cytochromes bl and a2 (sensitive to 2-n-alkyl-4-hydroxyquinol...

Artificial energy conservation in the respiratory chain. No native coupling site between cytochrome c and oxygen

“…However, coupling with TMPD is found with preparations from Mycobacterium phlei [23] and Azotobacter vinlandii [24]. We think that this discrepancy might reflect the different polarities of vesicular membrane preparations: the former would correspond to submitochondrial vesicles, while the later cases should resemble mitochondria.…”

Respiration‐linked proton translocation in Azotobacter vinelandii

“…Azotobacter vinelandii (NCIB 8660) was grown in batch culture at 30°C under high aeration, nitrogenfixing conditions as described previously [3,8]. Unless otherwise stated, cells were harvested either in the early stages of logarithmic growth (A6ao ~< 0.4, [02] < 150 #M; denoted excess oxygen cells) or during the post-logarithmic growth phase (A6so ~>2.5,[02] < 2/aM; denoted oxygen-limited cells).…”

“…cytochrome system (site III); the major cytochrome branch appears to be uncoupled [8][9][10]. Energy conservation at site I is absent from membranes prepared from cells grown at very high oxygen concentrations [8,11 ].…”

Oxidative phosphorylation in intact Azotobacter vinelandii

“…In the nitrogen fixing microorganism Azotobacter vinelandii the P/O ratios in cell-free extracts are low [2,3] . Furthermore, uncertainty exists about the number of phosphorylation sites [3] and the possible participation of a non-phosphorylating pathway [2].…”