Peter Nicholls

Complex formation by cytochrome c: A clue to the structure and polarity of the inner mitochondrial membrane

A reversible Michaelis complex between cytochrome c and cytochrome oxidase was postulated by Stotz [l] on kinetic grounds in 1938. As pointed out by Smith and Conrad [2], however, kinetic observations do not distinguish between an active ES complex and an inhibitory ES complex accompanied by an effective bimolecular reaction between free enzyme and free substrate. In the case of cytochrome oxidase this means that the complex of ferrocytochrome c with the oxidized form of the enzyme could be inert. Smith and Conrad [2] felt that the inhibition of the oxidase reaction by ferricytochrome c and by non-specific polycations favored the idea of an inert ES complex. Minnaert [3] derived various kinetic models, on the other hand, that incorporated a functional role for both active complexes with ferrocytochrome c and inhibitory complexes with ferricytochrome c. The physical existence of such complexes was demonstrated by Okunuki et al. [4] and by King and Takemori [5] . And Nicholls [6,7] brought forward three kinds of argument for an active role of these complexes in electron transport, showing that (i) the effects of ionic strength on enzyme turnover and apparent affinity for cytochrome c are best explained by assuming intramolecular electron transfer within the complex, that (ii) the rate of electron transport in the intact respiratory chain was consistent with the existence of such a complex in situ in the membrane and that (iii) strong analogies existed between cytochrome oxidase and cytochrome c peroxidase, both of which form tight complexes with cytochrome c and oxidize. it as a specific substrate, while non-specific peroxidases, which also cata-

Kinetic studies on the interaction of TMPD with cytochrome c and cytochrome c oxidase

Abstract The kinetics of the reduction of cytochrome c by ascorbate and ascorbate plus TMPD 3 have been examined. TMPD increases the reduction rate of cytochrome c by a factor of 30 relative to ascorbate alone. It is concluded that this is the principal reason why TMPD increases the activity of cytochrome c oxidase at a fixed concentration of cytochrome c and markedly diminishes the apparent K m value for cytochrome c . The reduction of cytochrome oxidase by ferrocytochrome c is identical in the presence or absence of TMPD (3.5 × 10 7 m −1 sec −1 ). The oxidation of TMPD in the absence of cytochrome c shows an average maximal turnover of two electrons (heme a ) −1 sec −1 when cytochrome a measured at 605 mμ is over 60% reduced. This identifies a slow rate of electron transfer between a and a 3 . It is suggested that in the presence of cytochrome c the reaction pathway may be directly from c to a 3 .

Cytochromes a and a3: Catalytic activity and spectral shifts in situ and in solution

Abstract 1. The inhibitory and spectroscopic effects of azide on phosphorylating and non-phosphorylating systems have been studied. In non-phosphorylating particles succinate oxidase inhibition requires 10 times the concentration of azide needed to inhibit the oxidase itself. 2. Reduction of cytochrome a in non-phosphorylating particles oxidizing succinate occurs at low azide concentrations; inhibition supervenes only if sufficient azide is present to cause appreciable steady-state reduction of endogenous cytochrome c . 3. The previously reported shift in the α band of reduced cytochrome a from 605 to 600 mμ in presence of azide is now observed in all respiratory states of the mitochondrion, in Keilin-Hartree particles and in the isolated oxidase. It is concluded that this form of cytochrome a is neither a high-energy compound nor a complex with azide but the result of heme-heme interaction between reduced a and the classical oxidized a 3 -azide complex. 4. The high sensitivity of mitochondrial respiration in ADP-stimulated State 3 to azide is not accompanied by an increased sensitivity of mitochondrial oxidase activity. The changes in steady-state reduction of cytochromes c and a parallel the inhibition of respiration. Relief of inhibition by uncouplers is not explained by changes in intramitochondrial azide concentration and does not involve the disappearance of cytochrome a with an α band at 600 mμ. 5. It is suggested that the relief of azide inhibition by uncouplers is due to a change in electron-transfer pathways permitting a direct reduction of a 3 by c and short-circuiting cytochrome a . 6. The spectrum of reduced a 3 is discussed and it is indicated that this cytochrome probably has no appreciable α band at 605 mμ but instead is characterized in the visible region solely by a broad band centered at 560 mμ.

Patterns of cytochrome oxidase inhibition by polycations.

Abstract1.The inhibition of cytochromec oxidase activity by three types of polycation (PL-3, PL-150 or 195, and salmine) § is described for three kinds of oxidase system: dispersed by Tween-80, detergent-free “soluble” oxidase, and particulate oxidase (submitochondrial particles).2.Salmine acts as a competitive inhibitor towards cytochromec in all three systems, with aKi between 1 and 4μM. PL-3 (low M.W. polylysine) acts as a non-competitive inhibitor of oxidation in all three systems, with aKi of between 10 μM (submitochondrial particles) and 100 μM (detergrent-free oxidase).3.PL-150 and PL-195, the high M.W. polylysines, act in three distinct ways, depending on the nature of the oxidase preparation: (a) as reversible competitive inhibitors, withKi of about 70 nM (with oxidase dispersed in Tween-80), (b) as stoichiometric inhibitors displaying pseudononcompetivie kinetics (with Keilin-Hartree submitochondrial particles), and (c) as “superstoichiometric” inhibitors, blocking up to 100 equivalents of oxidase, cytochromeaa3 (with detergent-free oxidase or with cholate-treated submitochondial particles).4.PL-195 also inhibits NADH and succinate oxidase activities in intact butc-deficient submitochondrial particles; sigmoidal inhibition curves can be observed in such systems. The rate of PL-195 binding was of the order of 106M−1 sec−1 and the true binding constant between 0.1 and 0.01 nM for the systems showing high affinity.5.High molecular weight polylysines may be useful in investigations of the topology and distribution of cytochromec binding sites on the mitochondrial membrane and in submitochondrial fragments and solubilized preparations. More than one oxidase molecule may be inhibited by one polymer molecule; submitochondrial fragments “opened” by cholate treatment may bind one polymer molecule to several surface sites; Keilin-Hartree particles, with a more “closed” configuration, sesm to bind one polymer molecule to a single site, suggesting a “ecrevice” structure.

Nematode biochemistry: XII. Mitochondria from the free-living nematode Turbatrix aceti

Abstract 1. 1. Biochemically active mitochondria have been isolated from the free-living nematode, Turbatrix aceti. Electron photomicrographs indicate that the preparation consists mostly of intact organelles. 2. 2. The mitochondria oxidized all of the tricarboxylic acid metabolites tested, as well as NADH. α-Phosphoglycerol, dihydroxyacetone, and palmityl-cocnzvme A were inactive. 3. 3. The mitochondria contain a functioning cytochrome system. Cytochrome a-a2, appears to be the terminal oxidase. 4. 4. Spectra of oxidized and reduced cytochromes are reported. 5. 5. An additional pigment is present in the mitochondria in 6-fold greater quantity than the other cytochromes. The material binds CO, and probably CN−. It is reduced by dithionite and, in the presence of CO, by succinatc or NADH. The function of this pigment is unknown. It does not appear to be a terminal oxidase.

Metabolism and enzymology of fluorosuccinic acids: II. Substrate and inhibitor effects with soluble succinate dehydrogenase

Abstract The interaction of a series of fluorosuccinic acids with soluble succinate dehydrogenase was examined. 2,2-Difluorosuccinate is converted to monofluorofumarate by the dehydrogenase with a Vmax about 0.25% and a Km approximately 15 times higher than succinate. The reaction is a nonoxidative elimination and is competitively inhibited by malonate to the same degree as the natural substrate. Di-, tri-, and perfluorosuccinic acids and mono- and difluorofumaric acids are all competitive inhibitors of succinate oxidation with respective K values of 4.0, 1.3, 1.1, 0.33, and 1.2 m m . Monofluorosuccinate gives a spectrum with the dehydrogenase similar to that produced by succinate. Difluorosuccinate and difluorofumarate give spectra that initially resemble those obtained with malonate or fumarate but change to resemble the spectrum obtained with malate or oxaloacetate. dl -Monofluorosuccinate is also oxidized by soluble succinate dehydrogenase with an apparent Vmax about 45% that of succinate and a Km approximately five times greater. Enzymatically generated l -monofluorosuccinate had a Vmax approaching that of succinate. The reactions of soluble succinic dehydrogenase with succinate and with monofluorosuccinate are treated according to a simplified version of the mechanism previously postulated by Zeylemaker et al. The enzyme catalyzed elimination of HF from difluorosuccinate is used as a model for an ionic hydrogen elimination mechanism occurring with the true substrates that reduce hydrogen acceptors.

Metabolism and enzymology of fluorosuccinic acids. I. Interactions with the succinate oxidase system

Abstract 1. 1. Both monofluorosuccinate and 2,2-difluorosuccinate are metabolized by Keilin-Hartree particles containing succinate oxidase and fumarate hydratase to give oxaloacetate as a final product. 2. 2. dl -Monofluorosuccinate is a substrate oxidized by the succinate oxidase system, with a vmax from 35 to 40% that of succinate and a Km of about 1.0 mM, slightly larger than that for succifinate. The product is monofluorofumarate. 3. 3. 2,2-Difluorosuccinate inhibits the oxidation of succinate by succinate oxidase in a competitive manner with a Ki of approx. 0.5 mM. It also slowly eliminates HF in the presence of the submitochondrial particles to give monofluorofumarate. 4. 4. Monofluorofumarate reacts in the presence of fumarate hydratase to give an unstable 2-fluoromalate which eliminates HF to give oxaloacetate. The production of HF was monitored by complex formation with yeast peroxidase; the production of oxaloacetate by inhibition of succinate oxidase activity and by its spectrum in the ultraviolet region. 5. 5. Oxaloacetate inhibition could be treated as if oxaloacetate were a competitive inhibitor of the particulate succinate oxidase with an apparent association rate constant of 6·103 M−1 · sec−1, a dissociation constant of 2· 10−3 sec−1 and an effective equilibrium constant of 0.3 μM at pH 7.4 and 25°. No evidence for any more complex binding process was obtained, although the occurrence of other steps during the reaction cannot be eliminated. 6. 6. The metabolic pathway followed by the fluorosuccinates explains their relative lack of toxicity compared with fluoroacetate, and the corresponding marked differences in toxicity between compounds (such as ω-fluoro odd-carbon fatty acids) which give fluorinated 3-carbon fragments and those (such as ω-fluoro even-carbon fatty acids) which give fluorinated 2-carbon fragments. 7. 7. The significance of the inhibitor and substrate relationships for various succinate analogs reacting with succinate dehydrogenase is discussed.