Phosphorylation coupled to NADH oxidation with fumarate in Streptococcus faecalis 10Cl

ARCHIVES

OF

BIOCHEMISTRY

AND

Phosphorylation

BIOPHYSICS

Coupled

137,

to NADH

Streptococcus I?. J. FAUST Microbiology

Section, Division Received

(1970)

Oxidation

faecalis

AND

of Biological November

392-398

with

Fumarate

in

1OCI’

P. J. VANDEMARK

Sciences, Cornell 5, 1969; accepted

University, January

Ithaca,

New York 1.4860

7, 1970

Phosphorylation coupled to the oxidation of NADH using fumarate as an electron acceptor was demonstrated in the cytochrome-free bacterium Streptococcus jaecalin lOC1. Cell-free extracts were separated into supernatant and particulate fractions by differential centrifugation at 48,000g for 25 min. The coupled phosphorylating activity was located primarily in the particulate fraction, where a P:O ratio of 0.19 wa= observed. The supernatant fraction contained nonphosphorylating oxidative enzymes and enzymes responsible for ATP formation in the absence of NADH. Oxidation and phosphorylation were inhibited by quinacrine HCl, bathophenanthroline disulfonate, and menadione. Malonate prevented NADH oxidation, presumably by inhibiting the enzyme fumarate reductase. Although this organism contains an active fumarate reductase, reverse electron transport could not be demonstrated with succinate as the electron donor and ATP as an energy source. A-CCP, a powerful uncoupler of oxidative phosphorylation, failed to uncouple this bacterial system. Uncoupling was observed with arsenite, and this was reversed with an excess of 2,3-dimercaptopropanol but not with 2-mercaptoethanol. High concentrations of rotenone, a site 1 inhibitor, and NO&NO, a site 2 inhibitor, reduced the P:O ratio. However, antimycin, another site 2 inhibitor, stimulated the observed P:O ratio by decreasing the level of oxidation. Since the level of phosphorylation remained constant, antimycin may be inhibiting a nonphosphorylating respiratory enzyme.

nature of respiratory transport and energy coupling not originally seen in the more complex mitochondrial systems. The demonstration of oxidative phosphorylation in the genus Streptococcus (ll13) is of particular interest since it involves a bacterium devoid of heme pigments. This organism, therefore, represents an unique system, uncomplicated by the presence of cytochromes, for the study of oxidative phosphorylation in the flavin region of the respiratory chain. Previous determinations of site 1 phosphorylation in mitochondria based on the difference between the P: 0 ratios obtained with NADH and succinate as substrates (14) are usually inaccurate. Other studies involving the substitution of various electron acceptors for oxygen, e.g., ferricyanide (15)) phanazine methosulfate (16)) Coen-

While studies of oxidative phosphorylation in bacteria are not so extensive as those employing mitochondrial systems, much of our present knowledge in this area is the result of bacterial investigations. Coupling factors and their role in oxidative phosphorylation were first demonstrated in bacteria (l-4). Similarly the investigation of high-energy intermediates formed during phosphorylation began in bacterial systems (5, 6). Although the role of quinones in the respiratory chain of mitochondria remains controversial (7), the function of napthoquinones in electron transport and energy generation in several bacterial species has been established by various workers (X-10). Therefore, these limited bacterial studies have provided important insights into the 1 This work was supported Service Grant FD00125.

b\- Public

Health 392

OXIDBTIVE

PHOSPHORYLATION

zyme Q1 (17), and fumarate (18, 19>, are not without drawbacks. Using ferricyanide and phenazine methosulfate as artificial electron acceptors, oxidative phosphorylation can often be observed in intact mitochondria but not in submitochondrial particles (20). Coenzyme &I would not be a suitable acceptor for studying systems which have a rotenone-insensitive site, such as Xaccharamyces cerevisiae, since the point of interaction of Coenzyme &I with the respiratory chain is difficult to ascertain (17). With the exception of its slow rate of reduction in mitochondrial systems (20), fumarate would seem to be an ideal acceptor for the study of site 1. While fumarate is not dissimilated by S. faecalis, it can serve as an alternate electron acceptor for this species (31). This paper describes the preparation and study of phosphorylation by cell-free extracts of X. faecalis during the oxidation of JSADH with fumarate as an electron acceptor. MATERIALS Culture

extracts.

grown in

conditions

METHODS

preparation

of cell;free

strain lOC1 was medium containing 1% tryptone, 0.5y0

Streptococcus

a extract,

AND and

faecalis

50 mM KsHPOb, 4 mM glucose, and 32 rnM potassium fumarate, with an initial pH of 8. The cells were incubated in l-liter volumes of medium for 10 hr at 37”. Anaerobic growth was maintained through the use of a Vaseline-paraffin seal. Cells were harvested by cent,rifugation at 11,OOOg for 15 min at 2’, washed once in 10 mM TES2 buffer, pH 7.4, and resuspended in 7 ml of TES. Cells were broken in an X-press (Biotech, New York, New York) at -25”. The broken cells were separated into supernatant and particulate fractions by centrifugation at 48,OOOg for 25 min, and the supernatant fraction was collected. The pellet was washed once by suspending in the TES buffer and recentrifuging at 48,OOOq for 20 min. The pellet was then resuspended in 3 ml of TES and centrifuged at 2000g for 10 miu to remove cell debris, and the milky-white supernatant, fluid then was collected as the particulate fraction. Measurement of NADH oxidation and coupled phosphorylation. The oxidation of NADH with yeast

2 The abbreviations used are: TES, N-h3 (hydroxymet,hyl) methyl-2-amino-ethanesulfonic acid; FI-CCP, trifluoromethoxycarbonylcyanide phenylhydrazone; NO&NO, 2 N-heptyl-4.hydroxyquinoline N-oxide.

IN STIZEPTOCOCCUS

faecalis

393

fumarate as the electron acceptor was followed spectrophotometrically at 340 nm in anaerobic cuvettes (Precision Cells, New York, New York). The reaction mixture consisted of 0.3 ml of stock Solution A (20) (which contained 6 rmoles of MgCl?, 1.4 pmoles of EDTA, 15 Mmoles of TES, pH 7.4, 9G @moles of 2-deoxy-n-glucose, 3.0 pmoles of ATP, 30 units of dialyzed hexokinase (ATP:Dhexose 6.phosphotrausferase, EC 2.7.1.1.), and 6 mg of dialyzed bovine serum albumin), 60 /*moles of “2Pi, pH 7.4 (approximately 1.7 X lo5 cpm per pmole), 13 rmoles of potassium fumarate, 375 pmoles of sucrose and 2-6 mg of cell extract protein ill the main cuvette, and 0.9 pmoles of NADH placed in the sidearm, with a total volume of 3.0 ml. The hexokinase was dialyzed overnight against 200-500 vol of 5 mM EDTA, pH 7.4, plus l’:& glucose. Bovine serum albumin was also dialyzed overnight against 200 vol of 10 mM Tris-sulfate, pH 7.4. To determine the level of endogenous phosphorylation, cont,rol cuvettes containing the above reaction mixture without NADH were run. The cuvettes were evacuated three times and flushed with nitrogen gas after each evacuation to obtain anaerobic conditions. The reactions were started by tipping the NADH from the sidearms and terminated after 3-5 min by the addition of 0.3 ml of 50yfi TCA to each cuvette. The reaction mixtures were then centrifuged, and glucose-6-32P was extracted with molybdic acid and isobutanolbenzene as described by Schatz and Racker (2). Protein was determined by the TCA precipitation method of Stadtman et al. (22). Reverse electron transport. Attempts to observe the succinate-linked reduction of exogenous NAD+ by the particulate fraction with ATP as an energy source (23) were run spectrophotometrically at 340 nm in anaerobic cuvettes. The reaction mixture contained 18 Mmoles of MgSOd, 15 pmoles of potassium fluoride, 15 @moles of succinic acid, 2 pmoles of NAD+, 150 rmoles of TES, pH 7.4, 540 pmoles of sucrose, 0.1 ml particulate fraction (containing 1.5 my protein), and 1, 10, or 40 pmoles of ATP added to the cuvette sidearm, with a final volllme of 3.0 ml. A control cuvette without ATP also was run. Both cuvettes were evacuated four times and flushed wit,h nit,rogen The reaction was initiated by addition of ,4TP from the sidearm. Chemicals. Bathophenanthroline disulfonate (sodium salt) and antimycin were graciously supplied by Dr. D. C. Wharton, and F,-CCP was donated by Dr. It. E. McCarty (Section of Biochemist,ry and Moleclllar Biology, Cornell University). All ot,her chemicals were obtained commercially.

394

FAUST

AND

VAN

DEMARK

RESULTS

Oxidation and phosphorylation by cell fractions. The levels of phosphorylation by supernatant, particulate, and combined supernatant and particulate fractions of S. faecalis during the oxidation of NADH with fumarate as the electron acceptor are shown in Table I. Despite the crude fractionation procedure consisting of differential centrifugation at relatively low centrifugal speeds (48,000g for 25 min), it is apparent that the location of oxidative phosphorylation is in the particulate or membrane fraction. Although there is strong NADH-oxidizing activity in the supernatant fraction, this activity does not appear to be coupled to phosphorylation. This bypass of the phosphorylation site by enzymes in the supernatant fraction appears analogous to the bypass enzymes observed in other bacterial systems (24). The level of phosphorylation was not stimulated but was decreased by the addition of supernatant to the particulate fraction. This again probably reflects the relatively crude fractionation procedure employed and the failure to isolate a supernatant-free particulate fraction. Figure 1 illustrates the relatively rapid oxidation of NADH by the particulate fraction of S. faecalis with fumarate as the electron acceptor. The low level of NADH oxidation in the absence of added fumarate is probably due to the failure to obtain complete anaerobiosis. Endogenous activity. Endogenous phosphorylation by the particles in the absence of NADH or fumarate (Table II) is relatively high, and it represents approximately 50 % of the total phosphorylation. The level of endogenous phosphorylation was highest in the supernatant fraction, which suggests that the endogenous activity detected in the particulate fraction may be the result of contamination by the supernatant fluid. Since the phosphorylating activity of X. faecalis appears to be located primarily in the particulate fraction whereas electron transport bypass and endogenous phosphorylation enzymes were mostly associated with the supernatant fraction, further studies on the nature of this phosphorylation were limited to the particulate fraction.

4 z?

c

0

1

2

3

4

Minutes

FIG. 1. Fumarate-dependent oxidation of NADH. The assay was performed as described in the text. The control (open circles) contained 0.4 ml water instead of fumarate (solid circles). Each cuvette contained 4.47 mg particulate protein. TABLE

I

COMPARISON OF NADH OXIDATION AND PHOSPHORYLATION COUPLED TO FUMARATE REDUCTION BY SUPERNATANT AND PARTICULATE FRACTIONV Fraction Supernatant Particulate Supernatant ticulate

+ par-

NADH G-6-9 oxidized' formed 106.24 44.67 69.47

3.66 8.62 3.52

P:O ratio 0.03 0.19 0.05

a The reaction was performed anaerobically as described in the text. The values have been corin a curected for endogenous 32P esterification vette containing all components except NADH (see Table II). b Expressed as nanomoles per minute per milligram of protein.

Electron transport inhibitors. The effects of various inhibitors and uncouplers of oxidative phosphorylation on S. faecalis can be seen in Table III. A relatively high concentration of F,-CCP (0.016 mM) failed to uncouple ATP synthe-

OXIDATIVE

PHOSPHORYLATION

IN

TABLE

II AND

Glucose-6-~~P formed(nmoles/min/mg protein)

Fraction

Supernatant Particulate

Complete system

Minus NADH

Minus fumarate

30.21 14.42

35.79 6.79

30.67 7.86

a The reaction was carried as described in t,he text.

out

anaerobically

TABLE EFFECT

OF INHIBITORS

Inhibitor

Complete system Fa-CCP Quinacrine HCl Bathophenanthroline Rotenone Menadione Malonate Antimycin

NOQNO

disulfonate

ON NADH

395

system, but did not lower the P:O ratio significantly. Antimycin, a classical inhibitor of electron transport near site 2 phosphorylation, decreased NADH oxidation with fumarate, yet the level of phosphorylation remained the same, therefore increasing the apparent P: 0 ratio. The inhibition of oxidation increased with higher concentrations of antimycin (up to 0.5 mM), thus raising the P: 0 ratio. Another electron transport and phosphorylation inhibitor for site 2, NO&NO, added in the same concentration as antimytin, very strongly inhibited both oxidation and phosphorylation. Reversal of arsenite uncoupling by dithiol reagents. As seen in Table IV, sodium arsenite uncoupled phosphorylation and stimulated oxidation. A 4-fold excess of 2,3-dimercaptopropanol in the presence of arsenite completely reversed the uncoupling effect. However, high levels of 2-mercaptoethanol did not alleviate uncoupling by arsenite. Reverse electron transport. The succinatelinked reduction of exogenous NAD+ by the particulate fraction was not observed in S. faecalis. Three concentrations of ATP were tried (see Materials and Methods) but

sis. Quinacrine HCL, an analog of flavin, partially inhibited oxidation and phosphorylation, and also lowered the P: 0 ratio. Bathophenanthroline disulfonate, which complexes ferrous ions, inhibited oxidation and phosphorylation in these extracts. High concentrations of rotenone (0.254 mM) inhibited NADH ox,idation and phosphorylation, whereas a lower level (0.02 mM) reduced phosphorylation with only a slight decrease in NADH oxidation. Menadione, an analog of napthoquinone, stimulated NADH oxidation and completely uncoupled phosphorylation. Malonate inhibited the reduction of fumarate about 50% in this

ENDOCENOUS ATP F~RM~T~~N IN NADH FUMARATE-FREE CONTROLS~

STREPTOCOCCUS fuecalis

III

OXIDATION

AND

PHOSPHORYLATION”

Final c~ncn (mu)

NADH oxidized*

G-6.31P formedb

P:O ratio

0.016 0.02 0.266 0.02 0.254 0.66 8.3 0.05 0.10 0.50 0.10 0.53

44.67 55.59 36.01 40.73 39.31 20.45 91.81 21.53 25.90 20.83 16.86 13.70 15.35

8.62 9.38 4.02 2.96 4.5G OC OC 3.93 9.09 7.97 8.15 OC 0.5W

0.19 0.17 0.11 0.07 0.12 0 0 0.18 0.35 0.38 0.48 0 0.03

a The reaction was performed anaerobically with fumarate and the particulate fraction as described in the text. All values were corrected for endogenous 32P esterification formed in a NADH-free control. Bovine serum albumin was omitted from the experiments with F,-CCP and menadione since it binds these inhibitors. b Expressed as nanomoles per minute per milligram of protein. c No phosphorylation detected above endogenous level. d In some experiments there was no phosphorylation.

396

FAUST TABLE

AND

IV

REVERSSL OF ARSENITE UNCOUPLING BY DITHIOL REAGENTS~ Arsenite (ITIM)

2,3-dimercaptopropanol bd

P-mercaptoethanol ha)

-

-

-

0.033 0.033 0.033

0.13 -

0.39

NADH oxidized’

G-6-32P formedb

P:O ratio

44.67 53.75 40.76 57.36

8.62 1.57 6.27 2.37

0.19 0.03 0.15 0.04

0 The arsenite and particulate enzyme fraction were incubated for 10 min at 4” in 0.25 M sucrose. The thiol reagent, prepared fresh daily, was then added to the medium, and incubation was continued for an additional 5 min. Finally, the mixture was added to the rest of the reaction components, as described in the text, and the experiment’was run anaerobically. Concentrations refer to the final reaction vessel. b Expressed as nanomoles per minute per milligram protein.

in no case was NAD+ reduced with succinate. DISCUSSION

S. faecalis has previously been reported to contain those respiratory components necessary for oxidative phosphorylation in the flavin region, e.g., flavoprotein-linked NADH dehydrogenase (reduced-NAD : (acceptor) oxidoreductase, EC 1.6.99.3) (25), nonheme iron (26), and napthoquinone (27). The action of various inhibitors and uncouplers in the present study provides evidence for the role of these components in the energy-coupled respiration of this species. Analogous to previous observations of NADH oxidation with aerobic extracts of this species (II), the partial uncoupling by quinacrine HCl (Table III) implicates flavins in the energy-linked oxidative pathway to fumarate. The studies of Butow and Racker (28) have previously implicated nonheme iron as a functional component of coupled phosphorylation in mitochondria. The inhibition of oxidation and phosphorylation by the iron-chelator bathophenanthroline disulfo-

VAN

DEMARK

nate suggests that nonheme iron is involved in oxidative phosphorylation by extracts of X. faecalis and lends support to the view that site 1 phosphorylation occurs at the level of nonheme iron. While the role of napthoquinone in the respiration of several bacterial species is well documented, evidence for its participation in the respiratory chain of S. faecalis is circumstantial and is based primarily on the observation that the respiratory chain of this species is labile to irradiation at 360 nm (25). In the present investigation menadione was found to completely uncouple phosphorylation, presumably by the bypass of energy-conserving components of the respiratory chain. It is interesting to note that menadione bears a structural similarity to 2-solanesyl-l , 4-napthoquinone which has been isolated from S. fuecalis (27). Uncoupling of phosphorylation by arsenite and restoration of this process by excess 2,3-dimercaptopropanol, but not by 2-mercaptoethanol (Table IV) indicates the involvement of a dithiol function in oxidative phosphorylation by X. faecalis cell extracts. Fluharty and Sanadi (29) observed a similar uncoupling effect in mitochondria by arsenite and attributed it to the formation of a cyclic thioarsenite compound. They later reported that organic arsenicals stimulate the ATPase as well as uncouple phosphorylation in a manner similar to another uncoupling agent, dinitrophenol (30). They localized the arsenite-sensitive site as being between the electron transport step and the oligomycin-sensitive step. Thus, the uncoupling action of arsenite seen in S. faecalis as well as in mitochondria suggests that a dithiol function is necessary for the energy-trapping reactions in oxidative phosphorylation. However the participation of sulfhydryl groups at other sites of the respiratory pathway cannot be ruled out. If the electron transport pathway of S. fuecalis is similar to that found in mammalian mitochondria, the respiratory components involved in the oxidation of NADH coupled with the reduction of fumarate could be illustrated in the following scheme:

OXIDATIVE

PHOSPHORYLATION

/

IN STREPTOCOCCUS ATP A

-c---cFp-FeNAD+

f

While the rotenone-sensitive component is not known, studies with mitochondrial systems (31) would indicate that it acts between nonheme iron and Coenzyme &I, or cytochrome b. Thus, b?. analogy one might speculate that it inhlblts S. j’uecalis respiration between nonheme iron and naphthoquinone. The inhibition of KADH oxidation by malonate presumably occurs at the fumaratc reductase (reduced NAD: fumarate oxidoreductase) step, since this enzyme in S. faecalis previously has been shown to be sensitive to malonate (32). The failure to observe energy-linked pyridine nucleotide reduction with succinate \vas not unexpected, since the phenomenon of reverse electron transport is not common in chemoorganotrophic bacteria (33). Furthermore, extracts of S. fuecalis, while possessing high fumarate reductase activity, are devoid of succinatc dehydrogenase (32). It has been demonstrated in several bacterial systems (34-36) that fumarate reductase and succinatc dehydrogenase (succinate: (acceptor) oxidoreductase, EC 1.3.99.1) are physiologically different enzymes. In addition to the information it provides concerning the nature of the respiratory chain in S. faecalis, the study of oxidative phosphorylation in this species should add to our understanding of the mechanism of site 1 phosphorylation. A comparison of the results of the present study with those of Sanudi and Icluharty (18) and Haas (19), who studied site 1 phosphorylation in submitochondrial particles with the fumarate assay, indicates that the properties of the systems are similar. Oxidation of KADH by fumarate in both the S. jaecalis and the mitochondrial systems is insensitive to the uncoupling action of CCP. Uncouplers,e.g., CCP, are believed to function by causing the dissipation of some high-energy intermediate, but do not act on so-called loosely coupled systems where the breakdown of this intermediate is extremely rapid. Therefore, the phosphorylation coupled to elec-

1 -NQ

Rotehone

jaecalis

397

Fumorate

- \ L[ Malbnate ‘Succinate

tron transport between NADH and fumarate would appear to be a “loosely coupled” system. In both systems the oxidation is inhibited by high levels of antimycin (Table III). While Haas found that in submitochondrial particles these levels of antimycin also interfered with phosphorylation, the level of phosphorylation in particulate extracts of X. jaecalis is unaffected. Since antimycin has been reported to prevent the structural change of the respiratory particle as well as its electron-transfer function (37), it is possible that in X. faecalis antimycin interferes with a nonphosphorylating respiratory enzyme such that only uncoupled respiration is inhibited. Oxidation and phosphorylation were inhibited in both systems by NO&NO, an inhibitor which is reported to be more effective with bacterial preparations than antimycin (3s). Since both antimycin and NOQNO are supposed to inhibit at site 2, i.e., between cytochrome b and cytochrome cl, their effect on these assays of site 1 phosphorylation in these non-cytochrome-containing bacterial particles would indicate that they act in at least two sites. The pattern of inhibitor effects on site 1 phosphorylation by submitochondrial particles using the CoQl assay (20) appears to differ from that observed with the fumarate assay. While both sJ;stems are inhibited by rotenone and menadlone, the NADH-CO&~ reaction is CCP-sensitive and antimycininsensitive. This CCP sensitivity would imply that the CO&~ assay involves a more tightly coupled system. The difference in sensitivity to antimycin of the two assay systems indicates that an antimycin-sensitlve site exists on the oxygen side of CO&~ but on the substrate side of fumarate. Since the Co&l assay is more abbreviated than the fumarate assay, this mav indicate that an additional component(s) is participating in the respiratory pathway with fumarate as the electron acceptor.

FAUST

AND

While the present investigation is only preliminary, it is apparent that further study of phosphorylation coupled to anaerobic electron transport by this cytochrome-free species would contribute to our knowledge of the comparative biochemistry of oxidative phosphorylation. ACKNOWLEDGMENTS The authors thank Dr. D. C. Wharton and Dr. G. Schatz, Section of Biochemistry and Molecular Biology, Cornell University, for their interest and advice. REFERENCES 1. PINCHOT, G. B., J. Biol. Chem. 206, 65 (1953). 2. TISSII~RES, A., AND SLATER, E. C., Nature London 176, 736 (1955). 3. BRODIE, A. F., AND GRAY, C. T., Biochim. Biophys. Acta 19, 384 (1956). 4. HOVENKAMP, H. G., Nature 184, 471 (1959). 5. RUSSELL, P. J., JR., AND BRODIE, A. F., in “Quinones in Electron Transport” (G. E. W. Wolstenholme and C. M. O’Connor, eds.), p. 205. Churchill, London (1960). 6. PINCHOT, G. B., Proc. Nat. Acad. Sci. U.S.A. 46, 929 (1960). 7. CHANCE, B., BONNER, W. D., JR., AND STOREY, B. T., Ann. Rev. Plant Physiol. 19,295 (1968). 8. BRODIE, A. F., AND BBLLANTINE, J., J. Biol. Chem. 236, 226 (1960). 9. ASANO, A., AND BRODIE, A. F., J. Biol. Chem. 239, 4280 (1964). 10. WHITE, D. C., J. Biol. Chem. 240, 1387 (1965). 11. GALLIN, J. I., AND VANDEMARK, P. J., Biothem. Biophys. Res. Commun. 17,630 (1964). 12. MICKELSON, M. N., Bacterial. Proc. 66, 139 (1968). 13. SMALLEY, A. J., JAHRLINCT, P., AND VANDEMARK, P. J., J. Bacterial. 96, 1595 (1968). 14. GREEN, D. E., BEYER, R. E., HANSEN, M., SMITH, A. L., AND WEBSTER, G., Fed. Proc. 22, 1460 (1963). 15. WEBSTER, G., J. BioZ. Chem. 240, 1365 (1965). 16. SMITH, A. L., AND HANSEN, M., Biochem. Biophys. Res. Commun. 8, 136 (1962). 17. SCHATZ, G., in “Methods in Enzymology” (R. W. Estabrook andM. E. Pullman, eds.),

VAN

DEMARK

Vol. X, p. 30. Academic Press, New York (1967). 18. SANADI, D. R., AND FLUHAR’L’Y, A. L., Biochemistry 2, 523 (1963). 19. HAAS, D. W., Biochim. Biophys. Acta 92, 433 (1964). 20. SCH.~TZ, G., AND ~~~~~~~~~ E., J. Biol. Chem. 241, 1429 (1966). 21. GU~S~LUS, I. C.; J. Bacterial. 64, 239 (1947). 22. ST~DTM~N, E. It., NOVELLI, G. D., AND LIPMANN, F., J. Biol. Chem. 191, 365 (1951). 23. ERNSTER, L., AND LEE, C., in “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, eds.), Vol. X, p. 729. Academic Press, New York (1967). 24. ASANO, A., AND BRODIE, A. F., Biochem. Biophys. Res. Commun. 19, 121 (1965). 25. DOLIN, M. I., Arch. Biochem. Biophys. 66, 415 (1955). 26. DOLIN, M. I., AND BAUM, It. H., Bacterial. Proc. 66, 96 (1965). 27. BAUM, R. H., AND DOLIN, M. I., J. Biol. Chem. 240, 3425 (1965). 28. Bvrow, R., AND RACKER, E., in “Non-heme Iron Proteins: Role in Energy Conversion” (A. S. Pietro, ed.), p. 383. Antioch Press, Yellow Springs, Ohio (1965). 29. FLUK~RTY, A. L., AND SANAUI, D. R., J. Biol. Chem. 236, 2772 (1961). 30. FLUHARTY, A. L., AND SANADI, D. R., Biochemislry 2, 519 (1963). 31. PULLMAN, M. E., AND SCHATZ, G., Ann. Rev. Biochem. 36, 539 (1967). 32. AUE, B. J., AND DEIBEL, R. H., J. Bacterial. 93, 1770 (1967). 33. GEL’MAN, N. S., LUKOYANOVA, M. A., AND OSTROVSKII, D. M., in “Respiration and Phosphorylation in Bacteria,” p. 152. PIenum Press, New York (1967). 34. WARRINGA, M. G. P. J., AND GIUDITTA, A., J. BioZ. Chem. 230, 111 (1958). 35. HIRSCH, C. A., RASMINSKY, M., DAVIS, B. D., AND LIN, E. C. C., J. Biol. Chem. 236, 3770 (1963). 36. JACOBS, N. J., AND WOLIN, M. J., Biochim. Biophys. Acta 69, 18 (1963). 37. RIESKE, J. S., BAUM, H., STONER, C. D., AND LIPTON, S. H., J. Biol. Chem. 242, 4854 (1967). 38. LIGHTBROWN, J. W., AND JACKSON, F. L., Biochem. J. 63, 130 (1966).