Mechanisms of Oxidative Phosphorylation— Observations and Speculation

MECHANISMS OF OXIDATIVE PHOSPHORYLATIONOBSERVATIONS AND SPECULATION GIFFORD B. PINCHOT* Plants use the energy from sunlight to convert carbon dioxide and inorganic materials into organic compounds, in this way conserving part of the light energy by converting it into chemical energy in the compounds formed in photosynthesis. All other forms of life, with the exception of chemosynthetic bacteria, get their energy by breaking organic compounds down again. This is done one step at a time with liberation of small amounts ofenergy at each step rather than all at once. This stepwise degradation makes it possible for living systems to conserve or retain much of theliberatedenergyin a biologically usefulform, rather than allow it to be wasted in the form of heat. The biological-energy-conserving reaction coupled to degradation is the formation ofadenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). In other words, some ofthe energy made available in breaking down organic compounds is used to convert low energy Pi to the comparatively high energy terminal phosphate ofATP. The ATP thus formed is the major source of energy for chemical, osmotic, and mechanical work in living organisms. ATP formation can be coupled to the energy liberated by aerobic or anaerobic breakdown of organic compounds. The aerobic or oxidative reaction is far more complete with liberation of much more energy per molecule ofstarting material. For example, in the anaerobic breakdown ofglucose to lactic acid, approximately 57 kilocaloriesofenergy are liberated per mole ofglucose, and two moles ofATP are formed. In the com- * McCollum-PrattInstituteand Department ofBiology,Johns Hopkins University. Contribution #433 from the McCollum-Pratt Institute and the Department of Biology, Johns Hopkins University . Thisworkwassupported by grantsfrom the U.S. PublicHealth Serviceand the National ScienceFoundation . TheabletechnicalassistanceofMr. MatthewHormanskiis gratefullyacknowledged. 180 Gifford B. Pinchot · Oxidative Phosphorylation Perspectives in Biology and Medicine · Winter 196$ píete oxidative breakdown of glucose to CO2 and water, over ten times as much energy is liberated, and over fifteen times as much ATP is formed. This process oflinking ATP formation to complete oxidation of substrate is called oxidative phosphorylation. It is an extremely important reaction in aerobic organisms, since it supplies roughly 90 per cent oftheir energy requirements. Phosphorylation coupled to oxidative degradation of substrates was discovered in the late 1930's by Kalckar [1] and Belitzer and Tsibakowa [2]. It soon became apparent from the work ofOchoa [3] that there were three phosphorylations perpair ofelectrons andhydrogen ions transported from substrate to oxygen over the so-called electron-transport chain. In other words, the P/O ratio was 3. This in turn meant that the substrate phosphorylation mechanism operating in glycolysis, in whichthe substrate itself is phosphorylated, was not an adequate explanation of oxidative phosphorylation since it could account for only one of the three phosphorylations observed. This in turn meant that there must be at least two phosphorylations between DPN (the first acceptor in the chain) and oxygen ; but Ochoa was unable to demonstrate them using reduced DPN (DPNH) as substrate. In 1949 Friedkin and Lehninger [4] were able to show that phosphorylation did indeed take place during the oxidation of DPNH. This observation then posed the problem of understanding the mechanism by which this very important energy-conserving series of reactions takes place. Oxidative phosphorylation had become one of the major challenges ofbiochemistry, and it attracted a number ofvery able investigators. It became apparent from the work ofa number ofpeople that there were three phosphorylation sites on the electron-transport chain. The first site ofATP synthesis was in the span between DPN and cytochrome b, the second between cytochrome b and cytochrome c, and the final one between cytochrome c and oxygen [4-9]. In spite of this elegant and precise work, very little was discovered about the actual mechanisms of the reaction because mitochondria, in which the reaction takes place in animals, proved until quite recently to be extraordinarily resistant to the usual biochemical approach offractionation and reconstitution . In 1951 Dr. Efraim Racker and I began to investigate oxidative phosphorylation in bacterial extracts in the hope ofovercoming this difficulty. Using extracts of Escherichia coli, a particulate and a soluble component 181 were separated, both ofwhich were necessary for the formation ofATP linked to the oxidation...