Lucile Smith

Composition and properties of the membrane-bound respiratory chain system of Micrococcus denitrificans

Abstract 1. The respiratory chain system of Micrococcus denitrificans closely resembles that of mammalian mitochondria; it includes cytochromes a + a 3 , two c -type and at least one b -type cytochrome and DPNH- and succinate dehydrogenases. All of the cytochromes are membrane-bound in preparations from aerobically grown bacteria. Ubiquinone is also localized in the membrane fraction. Although an o -type cytochrome may be present, there is no evidence that it is a functional part of the electron transport chain. The DPNH- and succinoxidase activities show sensitivities to inhibitors entirely similar to those of the mammalian system. 2. The DPNH- and succinate dehydrogenases appear to be distinct entities with different sensitivities to the kind and concentrations of ions in the reaction medium and to inhibition by treatment of membranes with detergent. Both dehydrogenases react with the same cytochrome system, and in most circumstances the dehydrogenases are the rate-limiting steps in the oxidation of the substrates. 3. M. denitrificans can modify the relative amounts of the different cytochromes present according to the conditions of growth; the content of b - and c -type cytochromes is higher and the a -type lower in cells grown anaerobically with nitrate than in cells grown aerobically. The rates of electron transfer are high in cells grown under all conditions tested. There is no evidence that the respiratory chain system is composed of fixed units with precise stoichiometry. 4. The DPNH- and succinoxidase activities of relatively intact membranes are stimulated markedly by freezing and thawing or exposure to sonic oscillation. Detergents can have both stimulatory and inhibitory effects. The data give evidence of the nature of orientation of the respiratory chain pigments on the membrane.

The effect of methemoglogin on the inhibition of cytochrome c oxidase by cyanide, sulfide or azide

Abstract Under our experimental conditions sulfide was a more potent inhibitor of a particulate preparation of cytochrome oxidase than was cyanide; azide proved to be a relatively weak inhibitor, all of which is in agreement with the observations of others. The undissociated species (H2S) appeared to be more inhibitory than the anionic species (HS−) in accord with the conclusions of others about HCN and HN3. Addition of methemoglobin to the oxidase inhibited by cyanide or sulfide restored the activity of the enzyme system, but the addition of methemoglobin to the azide-inhibited oxidase under the same conditions had little or no effect. Our results suggest that sulfide produces death in animals by inhibition of cytochrome oxidase, but such a mechanism seems unlikely in the case of azide.

CYTOCHROMES AND OTHER PIGMENTS OF BAKER'S YEAST GROWN AEROBICALLY AND ANAEROBICALLY.

Abstract Measurements of difference spectra and enzymatic activities of aerobically and anaerobically grown Saccharomyces cerevisiae show that: 1. 1. The yeast grown under both conditions synthesizes cytochrome pigments and flavoproteins which are rapidly oxidized on addition of oxygen and rapidly reduced when oxygen in solution is exhausted. The absorption spectra of the cythochromes of the two kinds of yeast are different. Although cytochromes α + α3 are missing from anaerobically grown yeast, there is evidence that another oxidase may be present. 2. 2. Both kinds of yeast contain active primary dehydrogenases, but they show different reactivities with added redox systems in anaerobically grown yeast, as compared with yeasts grown aerobically. 3. 3. The cytochrome system and flavoproteins which react rapidly with oxygen are associated with different kinds of cellular structures in the two kinds of yeast, and this may indicate differences in the structural arrangements of membrane-bound systems. 4. 4. Additional pigments are found in both aerobically and anaerobically grown yeast which show changes in absorption spectrum on addition or deletion of oxygen. However, these changes are very slow compared with those of the respiratory system. 5. 5. Both aerobically and anaerobically grown yeast synthesize a pigment with properties like one found in the microsome fraction of rat liver. 6. 6. Aerobically and anaerobically grown yeast synthesize comparable quantities of cytochrome c peroxidase (EC 1.11.1.5).

Measurements of ATP levels of intact Azotobacter vinelandii under different conditions

1. 1. The ATP level of intact cells of Azotobacter vinelandii grown on a nitrogen-free, minimal salts medium with mannitol as carbon source has been examined using the luciferin-luciferase method. Harvested, washed and starved cells had aerobic ATP levels similar to cells taken directly from the culture medium. 2. 2. The aerobic ATP level was always high and about the same with endogenous substrates or with added substrates giving a 10-fold variation of respiratory rates. On anaerobiosis, the ATP level fell to about 1/4 of the aerobic level. 3. 3. Under anaerobic conditions the rate of utilization of ATP was 0.4–0.6 and 1.6–2.3 nmoles/min per mg dry wt. at 12° with endogenous substrate and with added β-hydroxybutyrate or lactate, respectively. The rate of formation on addition of O2 to anaerobic cells in the presence of β-hydroxybutyrate was 12 nmoles/min per mg dry wt. When the latter was compared with the rate of O2 uptake with the same samples, P:O ratios of 2 were obtained (corrected for the rates of utilization). 4. 4. The aerobic ATP level was low in early log phase cells, then increased during growth to a maximum of 4.8–6.2 nmoles/mg dry wt. at the end of log phase growth. 5. 5. Aeration of anaerobic cells gave maximal ATP levels within about 30 sec at 12°, but linear O2 uptake continued for several minutes, until exhaustion of the O2 in the suspending medium. 6. 6. Oxidative phosphorylation in intact cells of Azotobacter vinelandii is an efficient process, but phosphorylation does not appear to be tightly coupled to O2 uptake.

The cytochrome system of Bacillus megaterium KM. The presence and some properties of two CO-binding cytochromes

Abstract 1. Difference spectra of Bacillus megaterium KM membrane preparations indicate the presence of two pigments which bind CO and which exhibit the spectral characteristics of cytochromes a 3 and o . Relative amounts of the pigments vary with growth stage of the organism, but both are present at all stages which have been investigated. The pigments are believed to be metabolically active because they are completely reducible by substrate (NADH) and are reoxidizable in the presence of air. CO difference spectra of whole cell suspensions are in agreement with spectra of the isolated membrane fragments. In particulate preparations and in whole cells, CO difference spectra suggest that the a 3 component binds CO much more readily than the o component; this behavior offers a possible explanation for the fact that cytochrome o has been detected in only a few other microorganisms, since CO binding is by definition the property used to identify this cytochrome. 2. A separation of the two CO-binding pigments is obtained by incubation of membrane preparations with pancreatic lipase. This treatment selectively removes the o pigment from the membrane, leaving the a 3 component associated with an enzymatically active particulate fraction.

The isolation and properties of the cytoplasmic membrane of Micrococcus denitrificans.

Abstract If harvested under certain conditions of growth, the walls of all of the cells of a culture of Micrococcus denitrificans are susceptible to digestion by low concentrations of lysozyme. This digestion apparently weakens the cell walls in such a way that osmotic lysis of the spheroplasts formed releases relatively intact cytoplasmic membranes, which can be collected by centrifugation at 30 000 × g for 45 min. Fragments of cell wall released are not sedimented under these conditions. The cytoplasmic membranes are similar to other cellular membranes in their dispersal by detergents and in their composition. Those from aerobically grown bacteria contain around 32 % lipid, 64 % protein and 0.6 % carbohydrate; membranes from cells grown anaerobically in the presence of nitrate have a slightly higher content of protein. Granules with properties similar to, but not identical with, those of poly-β-hydroxybutyrate found in other bacterial species can also be separated from the lysates of lysozyme-treated cells.

Interaction of macroions with the respiratory chain system of mitochondria and heart-muscle particles

Summary Studies have been made of the influence of salts and polyions on several activities of water-washed mitochondria and heart-muscle particles: cytochrome c oxidase (EC 1.9.3.1), NADH cytochrome c reductase and NADH oxidase, Polycations inhibit all enzyme activities involving reaction with soluble cytochrome c to about the same extent. The NADH oxidase activity of heart-muscle particles is not inhibited by amounts of the polycations which completely inhibit the other activities. The cytochrome c oxidase activity of the heart-muscle particles is very sensitive to the concentration of salts in the reaction medium, while the NADH cytochrome c reductase activity is relatively insensitive. The inhibition of the cytochrome c oxidase activity of water-washed mitochondria, but not of heart-muscle particles, increases on preincubation of the preparations with polycations and increases still more rapidly during turnover of the oxidase. Large quantities of cytochrome c are adsorbed by both mitochondria and heart-muscle particles at low ionic strength. There appears to be a rapid exchange between the adsorbed cytochrome c and the cytochrome c in solution. The data emphasize the differences in structure of the respiratory chain system of swollen mitochondria and small membrane fragments and show several aspects of the reaction of the endogenous cytochrome c of the mitochondrial system with neighboring members of the electron-transport chain.

Kinetics of the interaction of the cytochrome c oxidase of Paracoccus denitrificans with its own and bovine cytochrome c

We have devised a relatively simple method for the purification of cytochrome aa3 of Paracoccus denitrificans with three major subunits similar to those of the larger subunits of the mitochondrial cytochrome oxidase. This preparation has no c-type cytochrome. Studies were made of the oxidation of soluble cytochromes c from bovine heart and Paracoccus. The cytochrome-c oxidase activity was stimulated by low concentrations of either cytochrome c, providing an explanation for the multiphasic nature of plots of v/S versus v. Kinetics of the oxidation of bovine cytochrome c by the Paracoccus oxidase resembled those of bovine oxidase with bovine cytochrome c in every way; the Paracoccus oxidase with bovine cytochrome c can serve as an appropriate model for the mitochondrial system. The kinetics of the oxidation of the soluble Paracoccus cytochrome c by the Paracoccus oxidase were different from those seen with bovine cytochrome c, but resembled the latter if poly(L-lysine) was added to the assays. The important difference between the two species of cytochrome c is the more highly negative hemisphere on the side of the molecule way from the heme crevice in the Paracoccus cytochrome. Thus, the data emphasize the importance of all of the charged groups on cytochrome c in influencing the binding or electron transfer reactions of this oxidation-reduction system. The data also permit some interesting connotations about the possible evolution from the bacterial to the mitochondrial electron transport system.

Purification and properties of cytochrome c555 from a protozoan, Crithidia fasciculata

Abstract Cytochrome c 555 was isolated and purified from a protozoan, Crithidia fasciculata . This cytochrome exhibits a unique spectrum. In the reduced form, the maxima appear to have shifted 5 nm towards the red region in comparison to the mammalian mitochondrial cytochrome c 555 . In spite of this spectral difference, cytochrome c 555 is similar to the cytochromes c 550 in many respects. Both are cationic proteins and can be isolated and purified by essentially the same methods even though the former has an isoionic point at pH 8.8, while the latter at pH 10.0. Both have molecular weights around 13000 and have similar amino acid compositions except that cytochrome c 555 contains 1 to 2 residues of e - N -trimethyllysine per molecule of protein. Although the protozoans protein reacts with mammalian cytochrome oxidase, it reacts at a reduced rate compared with that exhibited by the cytochromes c 550 .

Effect of osmotic pressure of the medium on the volume of intact cells of Azotobacter vinelandii and on the rate of respiration

Abstract 1. 1. Addition of several salts, sucrose or glucose to starved washed cells of Azotobactor vinelandii resulted in a decrease of cellular volume and an increase in turbidity (light scattering). Glycerol caused no change in either. 2. 2. The changes in light scattering indicated that LiCl, NaCl and KCl did not penetrate the bacteria, but NH 4 Cl, MgCl 2 , MnCl 2 and CaCl 2 penetrated slowly. 3. 3. Measurements of packed cell volume along with dextran and sucrose impenetrable volumes in the presence of non-penetrating solutes showed that the cell as a whole decreased in volume as the medium osmolarity increased. There was no distinct plasmolysis evident. 4. 4. Concentrations of about 0.3 osM salts or sucrose had no effect on the rate of O 2 uptake on addition of mannitol as a substrate when the bacteria were suspended in 10 mM phosphate buffer. Higher concentrations inhibited the respiration and the extent of inhibition was dependent upon the osmolarity. The respiratory rate of cells suspended in 10 mM Tris-HCl buffer was less than that of cells in phosphate buffer, but it was also inhibited by concentrations of sucrose above 0.2 osM. The low respiration rate in Tris-HCl was stimulated on addition of low concentrations of salts, then higher concentrations of salts were inhibitory. The extent of stimulation and inhibition as a function of concentration varied with the different salts.