An X-ray structure of the F1 portion of the mitochondrial ATP synthase shows asymmetry and differences in nucleotide binding of the catalytic beta subunits that support the binding change mechanism with an internal rotation of the gamma subunit. Other structural and mutational probes of the F1 and F0 portions of the ATP synthase are reviewed, together with kinetic and other evaluations of catalytic site occupancy and behavior during hydrolysis or synthesis of ATP. Subunit function as related to proton translocation and rotational catalysis is considered. Physical demonstrations of the gamma subunit rotation have been achieved. The findings have implications for other enzymatic catalyses.

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An overview of research in the field of bioenergetics that led to the development of the binding change mechanism for ATP synthesis is presented, with emphasis on research from the author's laboratory. The text follows closely the Rose Award Lecture given at the 1989 meeting of the American Society for Biochemistry and Molecular Biology. Remarkable advances have revealed that the ubiquitous membrane-bound ATP synthase has unusual composition and properties. The enzyme complex has 1, 2, 3, or 9-12 copies of eight or more protein subunits. The catalytic sites are located on three copies of an approximately 55-kDa subunit. It has the strongest positive catalytic cooperativity known for any enzyme. Examples are given of selected experimental results that have provided insights into its mechanism. These include demonstration of the characteristics, location, and function of catalytic and noncatalytic adenine nucleotide binding sites and the incisive information provided by measurement of phosphate oxygen exchanges and distribution of 18(O) in ATP or Pi formed by catalysis. Research from various laboratories gives support to the binding change mechanism in which energy from proton translocation serves principally to promote release of tightly bound ATP, with sequential participation of three catalytic sites. Some speculative suggestions about a rotational catalysis and about the different forms assumed by the ATPase are included.

The Pi z HOH exchange reaction of oxidative phosphorylation is considerably less sensitive to uncouplers than the Pi T ATP and ATP ;± HOH exchanges. The uncoupler-insensitive Pi 2. HOH Previous findings have shown that the Pi T HOH exchange is less sensitive to 2,4-dinitrophenol than the Pi T. ATP exchange or the capacity for net oxidative l)hosphorylation (10)(11)(12). However, the significance or the source of this exchange has not been known. The possibility exists that it might reflect activities of enzymes such as alkaline phosphatase or pyrophosphatase known to catalyze a Pi ± HOH exchange (7), perhaps activated in some manner by 2,4-dinitrophenol. In addition, whether such behavior is limited to 2,4-dinitrol)henol or might be shown by more potent uncouplers of oxidative phosphorylation has not been shown. Results with other uncouplers and with oligomycin inhibition reported here cover these points.The effects of increasing concentrations of the potent uncoupler 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13) (13) on the exchanges catalyzed by mitochondria are shown ill Fig. 1. In the absence of uncoupler, the relative rates of the reactions Pi 2. HOH, ATP ¢± HOH, and Pi T ATP are about 12:6: 1, respectively, under the conditions used. At low concentrations of uncoupler, the Pi ± ATP and the ATP z HOH exchanges are much more sensitive to the uncoupler than the Pi z HOH exchange. At a concentration of S-13 sufficient to inhibit the Pi T ATP and ATP 2 HOH exchange by about 50%, the Pi T HOH exchange is inhibited by less than 5%. At a concentration S-13 that gives a near zero value for the Pi T ATP and ATP T HOH exchanges and a maximum value for the uncoupler-stimulated ATPase activity, the Pi T HOH exchange is still rapid and inhibited by only 35%. Responses similar to those reported in Fig. 1 for S-13 with mitochondria are also observed with 2,4-dinitrophenol and m-chlorocarbonvlcvanide phenylhydrazone.Important for the present considerations are the demonstrations, not given in detail here, of the effects of oligomycin.This antibiotic is a potent inhibitor of oxidative phosphorylation and inhibits the Pi T HOH exchange (14). In the absence of uncouplers and under conditions like those described with Fig. 1 Abbreviation: S-13, 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide.

1-Phosphohistidine and 3-phosphohistidine Phosphohistidine has been isolated as the solid lithium have been synthesized by reaction of histidine with salt and has been characterized by composition, ultraphosphoramidate. The two isomers have been sepaviolet and nuclear magnetic resonance spectra, titrarated by paper electrophoresis and anion-exchange tion curve, and rate of hydrolysis over a wide range of chromatography. The synthetic phosphohistidine isopH values. a-N-Acetyl-3-phosphohistidine, l-methylmer which is identical with the compound isolated from 3-phosphohistidine, a-AT-acetyl-1-methyl-3-phosphomitochondria and from a succinate thiokinase preparahistidine, and the isomers of phosphohistidine methyl tion has been identified as 3-phosphohistidine. 3-ester have been prepared and some properties reported.

These reflections present a perspective of how I and my graduate students and postdoctoral fellows, over a span of many years, arrived at the concept that ATP is made by an unusual rotational catalysis of the ATP synthase. A recent sketch of the structure of this remarkable enzyme is given in Fig. 1. Such a depiction is the culmination of the efforts of many investigators.1 The two portions of the enzyme are the membrane-imbedded F 0 and the attached F 1 that has three catalytic sites, principally on the large ␤ subunits. ATP is formed when protons pass through the F 0 , driving the rotation of the ring-shaped cluster of c subunits and the attached ⑀ and ␥ subunits. Other subunits attached to outer portions of the F 0 and F 1 served as a stator. The internal rotary movement of the ␥ subunit is coupled to sequential changes in the conformation of the catalytic sites. During ATP synthesis these conformational changes promote the binding of ADP and P i , the formation of tightly bound ATP, and the release of ATP.Revealing the mechanism of the ATP synthase became a major research goal in the latter part of my long career. This paper recalls how my career developed as related to the remarkable progress in biochemical knowledge. It presents the background and results of fruitful, as well as mistaken, approaches that were explored. The Early YearsBorn and educated through college in Utah, at the age of 21 I entered graduate school in the Department of Biochemistry at the University of Wisconsin in the fall of 1939. The biochemical research and teaching there were excellent. Not until years later did I appreciate all that is necessary to create such a fine scientific environment.I had had no previous courses or research experience in biochemistry and was uncertain about my career choice. By the end of my first year of graduate study the fascination of biochemical understanding and the addictive effect of experimental attempts to uncover new knowledge had firmly launched me toward a career in biochemical research. The Department of Biochemistry at Wisconsin was at the forefront of research in nutrition and metabolism. Recent achievements included the identification of nicotinic acid as a vitamin, the irradiation of milk to produce vitamin D, the discovery of a vitamin K antagonist (dicoumarin), and the discovery of lipoic acid as a growth factor for bacteria. At that time incoming graduate students were assigned to a mentor professor. Both Henry Lardy, from South Dakota, and I joined the group of Professor Paul Phillips whose major interest was in dairy cattle nutrition. Evidence had been obtained that vitamin C might help prevent reproductive difficulties in cattle, and one of my assignments was to find if vitamin C might ameliorate the reproductive failure that occurred in rats with vitamin E deficiency. No benefits of vitamin C were noted, but the rats 1 Except for a few instances, the mention of important advances in information about the ATP synthase and in related areas of biochemistry is included without specific ...