Biochemical mutants: an approach to mitochondrial energy coupling

Textbooks of biochemistry will explain that the otherwise endergonic reactions of ATP synthesis can be driven by the exergonic reactions of respiratory electron transport, and that these two half-reactions are catalyzed by protein complexes embedded in the same, closed membrane. These views are correct. The textbooks also state that, according to the chemiosmotic coupling hypothesis, a (or the) kinetically and thermodynamically competent intermediate linking the two half-reactions is the electrochemical difference of protons that is in equilibrium with that between the two bulk phases that the coupling membrane serves to separate. This gradient consists of a membrane potential term Δψ and a pH gradient term ΔpH, and is known colloquially as the protonmotive force or pmf. Artificial imposition of a pmf can drive phosphorylation, but only if the pmf exceeds some 150–170 mV; to achieve in vivo rates the imposed pmf must reach 200 mV. The key question then is ‘does the pmf generated by electron transport exceed 200 mV, or even 170 mV?’ The possibly surprising answer, from a great many kinds of experiment and sources of evidence, including direct measurements with microelectrodes, indicates it that it does not. Observable pH changes driven by electron transport are real, and they control various processes; however, compensating ion movements restrict the Δψ component to low values. A protet-based model, that I outline here, can account for all the necessary observations, including all of those inconsistent with chemiosmotic coupling, and provides for a variety of testable hypotheses by which it might be refined.

To examine the effect of O₂ and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes.

Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2′,7′-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O₂ tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1.

pH homeostasis was dependent on ROS. Na⁺/H⁺ exchanger (NHE) activity was inhibited at low O₂ tension (acid efflux reducing from 2.30 ± 0.05 to 1.27 ± 0.11 mM min⁻¹ at 1%). NHE activity correlated with ROS levels (r² = 0.65). ROS levels were increased by antimycin A (with levels at 1% O₂ tension increasing from 59 ± 9% of the value at 20% to 87 ± 7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red–green fluorescence ratio at 1% O₂ tension decreasing to 40 ± 10% of the value at 20%). The response to changes in O₂ and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A.

The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O₂ tension.

Functional mitochondria with respiratory control were isolated from the yeasts Saccharomyces cerevisiae and Schwanniomyces castellii. The presence of site I in Schw. castellii was indicated by higher ADP/O ratio than in S. cerevisiae where this site is absent. The ATPase V_max was higher in S. cerevisiae than in Schw. castellii mitochondria. The latter was increased by the DR12 nuclear mutation. Nevertheless, the stimulation by heat and the inhibition profile of oligomycins on mitochondrial F1–F0 ATPase activities were similar in all three tested strains. In S. cerevisiae and Schw. castelli wild type or mutant mitochondria, the well-known inhibition of F1–F0 ATPase activity by low concentrations of oligomycins is abolished at high inhibitor concentrations near 60 μg/ml suggesting uncoupling of F1 activity. At still higher oligomycin concentration the ATPase activity of both species and mutant is again strongly inhibited, suggesting an inhibitory effect on yeast F1 activity not detected so far.

This chapter discusses the clinical features, biochemistry, and molecular biology of the mitochondrial diseases. Various treatments are discussed, but this is still an embryonic field. Mitochondrial diseases have many causes, including mutations in nuclear and in mitochondrial genes encoding electron-transport chain (ETC) components, and in genes of intergenomic signaling. The disorders are caused by point mutations in the mitochondrial genome, but this may be an artifact of study as the mitochondrial genome is relatively small, and easily sequenced. These disorders affect mainly muscle and brain, although cardiac endocrine and other manifestations also occur. Various therapies for mitochondrial disease are pharmacologic and non-pharmacologic, and include physical and supportive therapies, metabolic therapies, and gene therapy. The mitochondrial disease diagnosis is very straightforward. It may simply involve mitochondrial DNA testing on a blood sample. In complex cases or unusual cases, diagnosis is probably one of the most demanding areas in modern medical practice and requires a close collaboration between the clinical and laboratory teams, interpreting results from different diagnostic areas.

The biochemical genetics approach is defined as the use of mutants, in comparative studies with the wild-type, to obtain information about biochemical and physiological processes in complex metabolic systems. This approach has been used extensively, for example in studies on the bioenergetics of the photosynthetic bacteria, but has been applied less frequently to studies of intermediary carbon and nitrogen metabolism in phototrophic organisms. Several important processes in photosynthetic bacteria — the regulation of nitrogenase synthesis and activity, the control of intracellular redox balance during photoheterotrophic growth, and chemotaxis — have been shown to involve metabolism. However, current understanding of carbon and nitrogen metabolism in these organisms is insufficient to allow a complete understanding of these phenomena. The purpose of the present review is to give an overview of carbon and nitrogen metabolism in the photosynthetic bacteria, with particular emphasis on work carried out with mutants, and to indicate areas in which the biochemical genetics approach could be applied successfully. In particular, it will be argued that, in the case of Rhodobacter capsulatus and Rb. sphaeroides, two species which are fast-growing, possess a versatile metabolism, and have been extensively studied genetically, it should be possible to obtain a complete, integrated description of carbon and nitrogen metabolism, and to undertake a qualitative and quantitative analysis of the flow of carbon and reducing equivalents during photoheterotrophic growth. This would require a systematic biochemical genetic study employing techniques such as HPLC, NMR, and mass spectrometry, which are briefly discussed. The review is concerned mainly with Rb. capsulatus and Rb. sphaeroides, since most studies with mutants have been carried out with these organisms. However, where possible, a comparison is made with other species of purple non-sulphur bacteria and with purple and green sulphur bacteria, and recent literature relevant to these organisms has been cited.

This chapter presents an overview on the mitochondrial (mt) genomes of ascomycetes, as in contrast to the mt genomes of mammals, there is a tremendous variability among genomes of this fungal group. This chapter illustrates that the present knowledge of the mitochondria1 (mt) genome is mainly based on two efforts: the complete sequencing of the human mt genome and the establishment of a genetic mapping system because of the various mt mutants in Saccharomyces cereuisiae. The mt genome is an ideal experimental system because of its small size. This chapter deals with the different principles of genome organization, the structure of genes—especially the mosaic genes—and the replication and transcription of these genomes. The proper function of the mt-encoded components is also dependent on their transport into the mitochondrion and the assembly with the nuclear components. Future research concentrates on the question of interaction between organelles, and between the organelles and the nucleus. Nuclear genes involved in mt functions have and can be studied in order to understand the complex interplay.