Manometric Uncertainties in Connection with Reports of High Phosphorus to Oxygen Ratios

The generally accepted maximal values for P:O ratios of mitochondria oxidizing cu-ketoglutarate, other diphosphopyridine nucleotide-dependent substrates, and succinate are 4, 3, and 2, respectively (1). In addition, the demonstration of three crossover points (2) in the electron transfer sequence has bolstered the concept of three sites of energy conservation in oxidative phosphorylation. Recently, however, Boyer (3) has suggested that P:O ratios greater than 3 are possible both on thermodynamic and on mechanistic grounds if the synthesis of adenosine triphosphate were linked to a 1-elect,ron transfer through each coupling site. Bieber (4, 5) has suggested a mechanism involving such a l-electron transfer, by which P:O ratios of 6 might be attained. Several authors have published P:O ratios exceeding the generally accepted values (6-11) ; recently Smith and Hansen (12) in this laboratory reported P:O ratios approaching 6 for heavy beef heart mitochondria with pyruvate-malate as substrate, and approaching 4 with succinate as substrate. P:O ratios greater than 3 were also reported by Smith and Hansen (12) for phosphorylating submitochondrial particles (13) with DPNH as substrate. Lynn and Brown (14) have observed high P:O ratios with rat liver mitochondria.’ In the manometric assays the experimental conditions used by the various authors were different, and the increased efficiency of phosphorylation was attributed to various factors; significantly, in most cases short incubation times were used and small amounts of oxygen were consumed (7, 8, 12).l An exception to the short assay periods and small amounts of oxygen consumed was the report of Beyer and Kennison (11) that gave P :0 ratios greater than 2 for the oxidation of succinate by liver mitochondria from vitamin K-deficient chicks. These authors suggested that such preparations might oxidize malate formed from the oxidation of succinate, leading to the formation of DPNH, the oxidation of which would result in P:O ratios greater than 2. In another report experimental details were not provided (6). We have undertaken a systematic study to determine which conditions