Horizons of Bioenergetics: Proceedings of a Symposium held at Bloomington, Indiana October 12-15, 1970

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SESSION A

MECHANISMS OF ENERGY CONVERSION

Outline

Chapter 1: CHAIRMAN’S REMARKS

Chapter 2: ATP FORMATION BY ANAEROBIC BACTERIA

Chapter 4: THE PRESENT STATUS OF THE RECONSTITUTION OF A FUNCTIONAL INNER MITOCHONDRIAL MEMBRANE

Chapter 5: A Ca2+ BINDING FACTOR AND OXIDATIVE PHOSPHORYLATION

Chapter 6: ROLE OF MEMBRANES IN ENERGY CONSERVATION

CHAIRMAN’S REMARKS

A.L. Lehninger,     Johns Hopkins University, Baltimore, MD

In the letter of invitation to this Symposium on Bioenergetics our hosts expressed the hope that our discussions would not become narrowly focussed or polarized. Rather, they wished us to attempt to synthesize some new concepts and to crystallize some basic questions amenable to experimental solution in the 1970’s.

To introduce our first session, which will be devoted to mechanisms of energy conversion, I should like to make some general observations on where we are today and to point out some directions of research from which we may envision new illumination arising.

There can be no doubt that the most central and overriding problem we face today in the study of photosynthesis and respiration is the nature of the chemical and physical events involved in the conversion of electron transport energy into phosphate bond energy. In both processes we have chains of electron-carrying proteins which are fixed in membranes, the structure of which is vital for their function. In both cases electron transport can lead not only to ATP formation but also to transport of ions such as H+ across the membrane, and to changes in the conformation of the membrane. We are faced today with a trilemma as represented by the three current hypotheses for the mechanism of energy conversion.

The chemical coupling hypothesis envisions that ADP is phosphorylated through a series of consecutive enzyme-catalyzed reactions having common chemical intermediates of a high–energy nature, similar in principal to the consecutive steps involved in the substrate-level phosphorylations of glycolysis, the only difference being that the enzymes concerned are fixed in a membrane rather than in solution in the cytoplasm. One or another of the high–energy chemical intermediates in this process is postulated to be utilized in bringing about transport of ions across the membrane and the characteristic changes occurring in the conformation of the membrane.

The chemiosmotic hypothesis postulates that electron transport occurs via a chain of geometrically oriented electron carriers and generates an electrochemical potential across the membrane, which is then used to drive the synthesis of ATP from ADP and phosphate by a physical coupling to a flow of electrical charges or of protons, rather than by a chemical coupling process via a common intermediate.

The conformational coupling hypothesis postulates that electron transport generates a high–energy conformational state of the electron carriers or of the whole membrane, which is then utilized to drive ATP synthesis. The intermediate high–energy state is postulated to involve the cooperative interaction of weak non-covalent bonds, such as hydrogen bonds or hydrophobic interactions. There are some who view the conformational coupling hypothesis as a special case of the chemical coupling hypothesis, the only difference being that conformational coupling utilizes as the energy-carrying intermediate a number of cooperative non-covalent bonds, rather than a single covalent bond.

As things stand today, both the chemical coupling and the chemiosmotic models can provide for most of the known properties of energy–conserving electron transport. But each has serious weaknesses. The chemical coupling hypothesis suffers from past failures to rationalize the necessity of membrane structure for oxidative phosphorylation and to prove the existence of high–energy chemical intermediates. Perhaps, however, in recent work in the laboratories of Drs. B. Chance and E. C. Slater we may see more tangible evidence for high–energy forms of the cytochromes. In its turn the chemiosmotic hypothesis has suffered from inadequate demonstration of the direct generation of gradients of protons or electrochemical potential across the membrane by electron transport. The conformational coupling hypothesis lacks a convincing molecular model which can serve as a prototype for the conversion of conformational energy into covalent bond energy. Even the important reconstitution experiments of Dr. E. Racker and his colleagues, significant as they are to experimental analysis of the componentry of oxidative phosphorylation, are unable to date to resolve this trilemma.

There may however be some other ways of looking at the problem. I believe much illumination is likely to come from consideration of the evolutionary and comparative aspects of the development and differentiation of energy–conserving redox systems. There are perhaps two major landmarks in biochemical evolution that are most relevant to our discussion on bioenergetics. The first, in my opinion, is the evolutionary jump that must have occurred when the first photosynthetic bacteria arose, possibly following a long period during which the first prokaryotes employed strictly anaerobic, fermentative energy–conserving mechanisms. This big jump later led, after the build-up of oxygen in the atmosphere, to the origin of aerobic bacteria, with membrane-linked energy–conserving electron carrying chains, and ultimately to the origin of eukaryotes. As we begin to acquire new insight into the background of these events, we might also derive some insight as to why the membrane-linked energy–conserving electron transport systems of photosynthetic and aerobic bacteria and of eukaryotes, arose from, or in preference to, the simpler, soluble substrate-level energy–conserving mechanisms characteristic of anaerobic bacteria. Very likely we still have important comparative lessons to learn from substrate-level energy–conserving mechanisms.

We should also ask whether ATP has always been the carrier of chemical energy between energy–conserving and energy-utilizing processes. Recent work of Drs. H. and B. Baltscheffsky for example, suggests that pyrophosphate may once have played such a role and may still do so in some organisms. But going back even further, it seems quite possible that there are still earlier energy-rich precursors of pyrophosphate groups. Here we may well give close consideration to recent experiments which suggest that respiration-linked active transport in bacteria does not require ATP or other phosphorylated intermediates and other experiments showing that Ca²+ transport in mitochondria is a process that takes primacy over oxidative phosphorylation. These considerations lead to the idea that perhaps the original function of membrane-linked energy–conserving electron transport systems was to preserve internal ionic and nutrient homeostasis by promoting active transport, and that the capacity to bring about phosphorylation of ADP was a later evolutionary development.

I think I have now raised more than enough issues and questions to get us off to a start. I should now like to introduce Professor H. A. Barker of Berkeley, to begin our Symposium with a discussion of energy conversion mechanisms in bacteria.

ATP FORMATION BY ANAEROBIC BACTERIA

H.A. Barker,     University of California, Berkeley, CA

Publisher Summary

This chapter highlights adenosine triphosphate (ATP) formation by anaerobic bacteria. Anaerobic bacteria make use of only a few energy-rich compounds as substrates for ATP-generating reactions. Diphosphoglycerate and phosphenol pyruvate, the phosphoryl donors of glycolysis and alcoholic fermentation, are used by a variety of anaerobic bacteria. The high-energy phosphoryl groups of diphosphoglycerate and the other phosphoryl donors are derived from inorganic phosphate by reaction with a precursor containing a suitable high-energy bond. The most frequently used phosphoryl donor for ATP synthesis in anaerobic bacteria is acetyl phosphate. The compound is formed from a wide variety of substrates by several different paths. A few anaerobic bacteria use carbamyl phosphate as a phosphoryl donor to ADP. Most of the ATP-generating reactions of anaerobes occur in aerobic organisms, although anaerobes frequently show a higher degree of specialization in the utilization of specific reactions. It is not yet certain that anaerobic bacteria catalyze any truly unique ATP-generating reactions, although this is still a possibility.

The work of Pasteur established the existence of bacteria that normally live in the absence of molecular oxygen. Since then a great number and considerable variety of obligate anaerobes have been described. Like aerobic organisms, these bacteria obtain energy for growth and other vital functions mainly by oxidation-reduction reactions. The reductants are mostly common organic compounds, or sometimes inorganic compounds, which are also used as energy sources by aerobic organisms. However, the oxidizing agents of aerobic and anaerobic organisms obviously differ, since by definition aerobes use molecular oxygen whereas anaerobes are generally restricted to the use of organic oxidants such as pyruvate, fumarate or glycine, although some of these bacteria can use inorganic oxidants, such as sulfate and carbon dioxide.

The difference in the oxidizing agents used by aerobes and anaerobes has an important consequence for the nature of the reactions involved in ATP generation. Most of the ATP available to aerobic organisms results from the oxidation of reduced pyridine or flavin coenzymes by molecular oxygen through the mediation of the cytochrome-containing electron transport chain. Most anaerobes cannot generate ATP in this way, since they lack both cytochromes and a high-potential oxidizing agent, like oxygen. Consequently they are usually dependent on substrate level phosphorylations to generate ATP. There are undoubtedly some exceptions to this generalization. The possible existence of several types of anaerobic electron transport phosphorylation is a subject of considerable current interest and controversy which I shall consider a little later.

SUBSTRATES FOR ATP-FORMING REACTIONS IN ANAEROBIC BACTERIA

substrate + ATP

substrate + ATP + X

First I wish to review briefly what is known about ATP generation by substrate level phosphorylations by anaerobic bacteria that ferment organic compounds.

A large number of fermentations using different substrates or forming different products are known. The substrates include most of the biologically important organic compounds including various types of sugars, simple and poly alcohols, amino acids, purines, pyrimidines, and various mono-, di- and tri-carboxylic acids. The products consist mostly of low molecular weight compounds, commonly including alcohols, ketones, fatty acids, hydroxy acids, carbon dioxide, hydrogen, methane, ammonia and hydrogen sulfide.

Although the variety of fermentation substrates and products is rather large, anaerobic bacteria make use of only a relatively few energy-rich compounds as substrates for ATP-generating reactions. These compounds are listed in the Table. Diphosphoglycerate and phosphenol pyruvate, the phosphoryl donors of glycolysis and alcoholic fermentation, are used by a variety of anaerobic bacteria that ferment carbohydrates by the Embden-Meyerhof pentose phosphate, or Entner-Doudoroff pathways or modifications thereof. It should be noted that the use of phosphoenol pyruvate does not actually result in a net formation of ATP since the phosphoryl group transferred to ADP was derived from another molecule of ATP at an earlier step in the fermentation. In contrast, the high energy phosphoryl groups of diphosphoglycerate and the other phosphoryl donors listed are derived from inorganic phosphate by reaction with a precursor containing a suitable high-energy bond.

The most frequently used phosphoryl donor for ATP synthesis in anaerobic bacteria is probably acetyl phosphate. This compound is formed from a wide variety of substrates by several different paths. In carbohydrate fermentations by heterofermentative lactic acid bacteria, acetyl phosphate is formed directly from xylulose-5-phosphate by the phosphoketolase reaction. More commonly, acetyl phosphate is formed from acetyl coenzyme A by the phosphotransacetylase reaction. Acetyl CoA may be formed by the oxidation of pyruvate or acetaldehyde, or by the thiolytic cleavage of the coenzyme A derivative of a β-keto acid. These acetyl CoA precursors can be derived in turn from a number of fermentable substrates including carbohydrates, amino acids, organic acids and alcohols.

The other acyl phosphates listed refer to propionyl-and butyryl-phosphates and other acyl phosphates formed in anaerobic oxidations of branched chain and aromatic amino acids, such as leucine and tyrosine. It is probable that each of these compounds can serve as a phosphoryl donor to ADP in some organisms. A kinase has been purified from Clostridium butyricum that utilizes propionyl- and butyryl-phosphates rapidly, and isobutyryl- and valeryl phosphates more slowly (Twarog and Wolfe, 1962). An enzyme catalyzing the formation of these acyl phosphates from the corresponding coenzyme A derivatives is also present in Clostridium butyricum and several other Clostridia (Valentine and Wolfe, 1960). Relatively little is known about the kinases and phosphotransacylases acting on other acyl phosphates.

A few anaerobic bacteria use carbamyl phosphate as a phosphoryl donor to ADP. This has been shown most clearly with Streptococcus faecalis which can use arginine as a supplementary energy source (10−1) is suitable for ATP synthesis. Bauchop and Elsden (1960) have shown that ATP formed in this way is used efficiently to increase the growth of Streptococcus faecalis when other essential nutrients are present in excess. There is some evidence that S. faecalis can also use agmatine, the decarboxylation product of arginine, to form ATP by an analogous series of reactions (Deibel, 1964).

Figure 1 ATP FORMATION FROM ARGININE IN S. faecalis

Another example of the use of carbamyl phosphate as an ATP precursor is provided by the studies of Valentine, Wolfe and their associates (Valentine and Wolfe, 1960; Valentine et al., 1962) on the fermentation of allantoin by Streptococcus allantoicus. This bacterium converts allantoin by several steps to carbamyl oxamate which undergoes a phosphorolytic cleavage to carbamyl phosphate and oxamate (Fig. 2). The energetics of this reaction are favorable for carbamyl phosphate formation (Bojanowski et al., 1964). The reaction probably provides much or all of the high-energy phosphate derived from the fermentation of allantoin.

Figure 2 ATP FORMATION FROM ALLANTOIN BY S. allantoicus (Valentine et al., 1962)

There is some evidence that creatine and creatinine can be utilized as another source of carbamyl phosphate by anaerobic bacteria (Sjulmajster, 1960), but the immediate precursor of carbamyl phosphate, presumably carbamyl sarcosine, apparently has not been characterized. It is also possible, but not yet demonstrated, that carbamyl phosphate may be formed during the fermentation of some pyrimidines.

The last of the known substrate-level ATP-generating reactions is that catalyzed by the formyltetrahydrofolate synthetase of purine-fermenting bacteria (Fig. 3) (Rabinowitz, 1960; Himes and Rabinowitz, 1962). This enzyme couples the cleavage of 10-formyl THF to formate and tetrahydrofolate with the phosphorylation of ADP. Formyl-THF is derived from xanthine or other purine by a complex series of reactions in which formiminoglycine is the key intermediate. This compound reacts with tetrahydrofolate to give the formimino derivative, which is then converted in two steps to 10-formyl-THF, the immediate precursor of ATP. Only one purine-fermenting Clostridium, Clostridium cylindrosporum, is presently believed to use this ingenious series of reactions as a major path of ATP generation. Even closely related purine-fermenting Clostridia appear to use the carbon of the formimino group, after reduction, mainly for the synthesis of serine from glycine. Serine is then converted via pyruvate and acetyl CoA to acetyl phosphate, a more conventional source of high-energy phosphate.

Figure 3 ATP FORMATION FROM FORMYL-THF IN XANTHINE FERMENTATION BY Clostridium cylindrosporum (Himes and Rabinowitz, 1962)

Although the evidence for ATP generation from formyl-THF is rather convincing for only one organism, there are some indications that formyl-THF may he used in this way in the fermentation of methanol by methane-producing bacteria (Stadtman, 1967; Wood et al., 1965). It is also theoretically possible to generate ATP from formiminoglutamate, a probable intermediate in the fermentation of histidine by some Clostridia (Wachsman and Barker, 1955), but this has not yet been demonstrated.

ATP formation in anaerobic bacteria can generally be attributed to one of the substrate-level phosphorylation reactions I have mentioned. However, a few anaerobic processes are known in which ATP formation cannot easily be explained by substrate level phosphorylation and in which electron transport phosphorylation has been postulated. These include the ethanol-acetate fermentation by Clostridium kluyveri, the reduction of glycine by Clostridium sticklandii, and the processes of sulfate and carbon dioxide