Bacterial Ferredoxin' R

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BACTERIOLOGICAL REVIEWS Vol. 28, No. 4, p. 497-517 December, 1964 Copyright @ 1964 American Society for Microbiology Printed in U.S.A. BACTERIAL FERREDOXIN' R. C. VALENTINE Department of Biochemistry, University of California, Berkeley, California

INTRODUCTION ...... 497 Low-Potential Electron Transport...... 498 ISOLATION OF FERREDOXIN FROM BACTERIA ...... 500 Purification ...... 500 Assays...... 500 Phosphoroclastic Assay ...... 501 Hydrogenase Assay ...... 502 Other Assays ...... 502 Distribution...... 502 PROPERTIES OF CRYSTALLINE FERREDOXIN ...... 503 Oxidized and Reduced States ...... 503 Spectra ...... 504 Composition ...... 504 ENZYMATIC REDUCTION OF FERREDOXIN ...... 505 Phosphoroclastic System ...... 505 a-Ketoglutarate ...... 506 Dithionite as H2 Precursor ...... 507 Hypoxanthine ...... 507 Coupling with Formate...... 507 OXIDATION OF REDUCED FERREDOXIN ...... 507 Urate ...... 507 Nitrite and Hydroxylamine ...... 508 Reduction of CO2 and Carbon Compounds ...... 509 FERREDOXIN-LINKED PYRIDINE NUCLEOTIDE REDUCTIONS ...... 509 Korkes Factors ...... 509 H2-NADP ...... 509 Pyruvate-NADP ...... 510 Ferredoxin-NAD System ...... 511 FERREDOXIN AND BIOLOGICAL NITROGEN FIXATION ...... 512 Ferredoxin Requirements for Nitrogen Fixation ...... 512 COENZYME NATURE OF FERREDOXIN ...... 512 Coenzyme for Reductions and Oxidations ...... 512 PHOTOSYNTHETIC FERREDOXIN ...... 514 Chromatium...... 514 Comparison with Clostridia ...... 514 Ferredoxin Versus Photosynthetic Pyridine Nucleotide Reductase ...... 515 LITERATURE CITED...... 515 INTRODUCTION protein was named ferredoxin by members of the Amber-colored extracts of Clostridium pas- DuPont biochemistry group who discovered it teurianum have yielded a new biological electron (20). Ferredoxin has the unique property of carrier (Fig. 1). This brown iron-containing being the most electronegative electron carrier yet found in the oxidation-reduction chain in 1 The word ferredoxin was coined by D. C. Wharton of the DuPont Co. and applied to the the chemical structure of the molecule; the pros- "iron protein" first obtained from Clostridium thetic group and catalytic function of iron and pasteurianum (20). Compounds from a variety of sulfur are not known. All ferredoxins display simi- bacteria and photosynthetic tissues have since lar biological activities but show varying degrees been called ferredoxins. Formal classification of of chemical differences; a useful classification ferredoxin-like compounds has not been attempted must, therefore, await further chemical charac- in this review because of insufficient knowledge of terization of ferredoxins from different sources. 497 498 VALENTINE BACTERIOL. REV. C"t **: .:.. : A.: .:.

.. ; c R lo * .... *...... :....,.#..

...... A;. #

OF: w .. :. .::::...... :.:...;.::...... :::...... ::...... : A.:f..,__f

C. cy/indrosporum

FIG. 1. Photomicrograph of ferredoxin crystals from several clostridia [courtesy, W. Lovenberg, B. B. Buchanan, and J. C. Rabinowitz (18)].

bacteria. Ferredoxin from C. pasteurianum has a electrons activated by light energy during the redox potential (E'o) of -417 mv at pH 7.55 photochemical act in the leaf (3, 33). (33). Ferredoxin functions as an electron-mediating catalyst for the biological production or Low-Potential Electron Transport utilization of hydrogen gas by bacteria (20, 21, Research on bacterial ferredoxin has helped to 35, 40); a related compound has been isolated clarify a useful concept: low-potential electron from spinach leaves and functions in photosyn- transport. A biological redox scale for ferredoxin thesis as an electron-trapping agent for the and several other precursors of molecular hydro- VOL. 28, 1964 FERREDOXIN 499 gen is shown in Fig. 2; pyruvic acid and "excited XH Ferredoxin YH chlorophyll" are at the top of the scale, and low- (oxidized) potential cytochrome c3 is at the bottom. The redox potential of the "excited chlorophyll" is (A) (B) speculative but is thought to be equivalent to or lower than the hydrogen electrode (3). Ferredoxin X Ferredoxin Y serves as an oxidation-reduction catalyst trans- (reduced) ferring electrons from the low-potential donors to electron-accepting compounds such as pyridine FIG. 3. Low-potential electron-transport chain re- nucleotides (see scale). A generalized low-poten- quiring ferredoxin. tial electron-transport chain involving ferredoxin is shown in Fig. 3. In this scheme, electrons from also be a a-ketoglutarate, H2, hypoxanthine, an electron donor such as pyruvic acid (repre- formate, dithionite, or the electrons activated by sented by XH) are passed to oxidized ferredoxin light energy during photosynthesis (33, 40). In with the aid of a specific dehydrogenase (A). addition to its role in biological hydrogen-evolv- Oxidized ferredoxin (dark brown in color) is ing reactions, ferredoxin is the catalyst for a converted to reduced ferredoxin (colorless) wide variety of reactions in which hydrogen gas during the reaction. Colorless ferredoxin donates or pyruvate serves as reductant for synthesis of its electron to the next member of the chain (Y). cellular constituents; one of the most important Interaction of colorless ferredoxin and Y [again of these is N2 fixation. Recently, Mortenson (22) a specific reductase (B) is required] yields reduced showed that ferredoxin serves as one of the Y (YH) and oxidized ferredoxin. In this manner, catalysts for biological nitrogen fixation. Mor- electrons flow from the electron donor -- ferre- -+ tenson (22) suggested that ferredoxin functions doxin acceptor. as an electron-transport coenzyme for the nitro- In a similar manner, many hydrogen-producing gen reductase system (nitrogenase)-perhaps bacteria oxidize ferredoxin by the hydrogenase mediating electrons directly to nitrogen reduc- reaction and evolve large quantities of hydrogen tase. Ferredoxin also mediates the reduction of gas (20, 39). A widely occurring keto acid, such nitrite ion to ammonia, an important reaction as pyruvic acid, serves as precursor of hydrogen in the nitrogen cycle of plants (17, 40). gas and is a good example of the electron donor Among the bacteria, the clostridia possess a (XH) shown in the scheme (12, 20, 21). XH may highly developed system for low-potential transport and have proven to be useful tools for -0.6 V research in this area. Gest (7) earlier recognized .-_ the value of the clostridial system for studies on low-potential transport, as evidenced by his Excited generalization concerning the photoproduction chlorophyll ', -C H3COCOOH of H2 by photosynthetic bacteria and H2 produc- - (a2-keto acids) tion by clostridia: _Mlethyl viologen "It is likely that photoproduction of H2 has an important significance for the mecha- H2 - Ferredoxin nism of electron transport in all types of HCOOH photosynthetic reactions. From the very Hypoxonthine - -B(enzyl viologen existence of light dependent H2 evolution, it may be inferred that the photochemical TPN DPN generation of electrons (reducing power) occurs in a reaction characterized by a redox potential well below those of the pyridine - Cytochrome-C3 nucleotide coenzyme systems and, accord- ingly, that these coenzymes are probably not -0.2 V reduced by "primary" acts. In this connec- FIG. 2. Redox scale for low-potential compounds tion, it is tempting to speculate that the (value is speculative for excited chlorophyll). carriers involved in the early stage of electron 5-00 VALENTINE BACTERIOL. REV.

transport in photosynthesis may be similar mary of the methods used by Lovenberg, Bu- to those participating in the phosphoro- chanan, and Rabinowitz (18) for crystallization elastic reaction of the clostridia..." of ferredoxin from C. pasteurianum, C. acidiurici, C. butyricum, C. tetanomorphum, and C. ISOLATION OF FERREDOXIN FROM BACTERIA cylindrosporum is shown in Table 1. Purification Assays Preparations of ferredoxin are easily made from cells of several different species of bacteria Purification of bacterial ferredoxin was (5, 18, 20, 23, 33, 35); a common starting ma- achieved only after development of suitable terial is a water extract of C. pasteurianum (20, assays. These assay procedures were similar to 33). Methods for purification and crystalliza- the hydrogenase assay described by Peck and tion of ferredoxin have been described by several Gest (27), in which the low-potential dyes methyl authors, and details of these fractionations will and benzyl viologen served as electron donors not be given here (5, 18, 20, 23, 33). All workers for hydrogen evolution. It is of interest to outline make use of the specific adsorption of ferredoxin some of the "dye" experiments of Peck and on diethylaminoethyl (DEAE)-cellulose columns Gest (27) which led to the isolation of ferredoxin: for their purification schemes. Ferredoxin is "Methyl viologen is one of a family of readily separated from most cellular proteins dyes, described by Michaelis and Hill (1933) and is isolated as a coffee-colored band (Fig. 4A) [reference 19 in this paper], with the general adhering to the top of a column of DEAE cellu- structure shown in Figure 1 [Fig. 5 in this lose (20, 35). Purification of ferredoxin by DEAE paper]. cellulose (Fig. 4B) and Sephadex column chro- "These indicators differ from most other matography and ammonium sulfate fractiona- oxidation-reduction dyes in several impor- tion yields a highly purified form of ferredoxin: tant respects. They are colorless in the oxi- crystalline aggregates which appear as fine brown dized state, while the reduced forms exhibit needles or rosettes (Fig. 1) (5, 18, 33). A sum- a deep blue or violet color. The stable .A. r :.3 B .. .R.. '.. *:: ': :......

.. a) *:: a) 0 __E Q. -c C: 0a. .T_ cn _s.,; ,j, 0 :,. 0 .'' -o ::e CL 3._ *: .....',.. 0._ To *: .::;. so .:.: '!. C:

:::: D ::

-J0 0 5 10 15 20 4!i:A Tube number FIG. 4. (A) Ferredoxin band on DEAE cellulose (courtesy, R. S. Wolfe). (B) Elution diagram offerredoxin from DEAE cellulose (35). VOL. 28, 1964 FERREDOXIN 501

TABLE 1. Purification of clostridial ferredoxin (5, 18)*

Source

C. C. Fraction C. C.pasleurianumpsteriaumCC. acidiriciadittici C. butyricumbtyrcum tetanomorphum cylindrosporum Units! A3s0/ Units/ A3so/ Units/ A3so/ Units/ Amso/ Units/ As!o/ mg Asot mg A28o mg As2g mg A280 mg A280

Sonic extract ...... 0.4 - 0.8 - 0.5 0.3 - 1.1 First DEAE-cellulose column ..... 10 0.29 8 0.08 19 0.03 7 0.13 5 First Sephadex G-25 column ...... 13 0.50 15 0.14 78 0.06 22 0.20 18 0.03 Second DEAE-cellulose column ... 18 0.78 27 0.21 58 0.37 35 0.71 24 0.17 Crystalline ferredoxin ...... 67 0.82 54 0.78 100 0.83 51 0.83 94 0.80 * The procedure used for the preparation of each fraction was similar to that previously described for Clostridium acidiurici ferredoxin (5, 18). t Ratio of absorption at 390 and 280 mg.

Oxidized form Reduced form and colloidal Pd as the catalyst. Assuming that hydrogenase acts catalytically in a R R similar manner, formation of H2 from a +1+ reservoir of reduced methyl viologen would HC CH HCN CH be anticipated." UH ICOH Peck and Gest (27) found that bacterial hy- C C drogenase rapidly removed the single electron from reduced methyl viologen, yielding hydrogen gas. Subsequently, a wide variety of hydrogen- HOC CH HOC COH evolving or hydrogen-utilizing reactions by bacteria were shown to be strongly stimulated 'N' R= Benzyl or N by methyl viologen. Mortlock, Valentine, and R methyl group R Wolfe (24) reported that pyruvate oxidation to FIG. 5. Structure of viologen dyes (27). H2 by an enzyme fraction from C. butyricum required methyl viologen, and Whiteley and Ordal reduced form under physiological conditions (44) observed that hydrogen formation from (pH < 12) is generated by the addition of hypoxanthine by Micrococcus lactilyticus was one electron. At 30 C, the normal potential stimulated by methyl viologen. More recently, of methyl viologen is -0.446 volts, and Tagawa and Arnon (33) used methyl and benzyl the potential of the system is independent of viologen as nonphysiological carriers for the pH (Michaelis and Hill, 1933) [reference reduction of nicotinamide adenine dinucleotide 19 in this paper]. Since the potential of the phosphate (NADP) by chloroplasts in the light hydrogen electrode decreases with increas- and in the dark. All of these experiments focused ing pH, the potential of the dye system under attention on a "natural" low-redox carrier and alkaline conditions is more positive than have served as a guide for development of that of the hydrogen electrode, whereas in assays for ferredoxin. acid solution it is more negative than that of the hydrogen electrode at the same pH. Phosphoroclastic Assay It would be expected, therefore, that molecu- In our first experiments, ferredoxin was lar hydrogen would be evolved in acid solu- recognized as a brown protein which stimulated tion from the reduced form of the dye in acetyl phosphate production from pyruvate with the presence of a suitable catalyst such as a DEAE-treated clastic extract (20, 35). H2 palladium or platinum. Such a reaction was evolution and acetyl phosphate production from observed by Michaelis and Hill (1933) [ref- pyruvate were completely abolished by passage erence 19 in this paper], who used chromous of crude extracts of C. pasteurianum through a chloride as the reductant for methyl viologen small DEAE-cellulose column (20, 35). In this 502 VALENTINE BACTERIOL. REV.

A Fd + Pi - .x- Acetyl -P + ± Pyruvate CoA, TPP CO2, H2 E 3 0

o I- ) o E ,Ferredoxin-free clostic enzymes 0 -1 0100 .Lmoles sodium pyruvote I ,lOOLmoles potassium phosphate (-) buffer, pH 6.5 -30 units CoA 0.02 0.04 0.06 0.08 as desired *Ferredoxin mg Ferredoxin added FIG. 6. (A) Clastic test tube assay for ferredoxin (20). (B) Ferredoxin concentration versus acetyl phosphate production with the clastic assay (29). manner, two fractions were easily obtained-the Other Assays acidic ferredoxin molecules were held back by Ferredoxin shows a wide absorption maximum the column (see Fig. 4A), while a mixture of in the visible region at about 390 myt, as do other ferredoxin-free elastic enzymes, including hydro- colored compounds found in crude bacterial genase, pyruvic dehydrogenase, transacetylase, extracts. This greatly hinders direct spectral and other enzymes, was not tightly bound and determination of ferredoxin in extracts; the passed directly through the column. Pyruvate widely used clastic assay, on the other hand, cleavage by this DEAE-column eluate was re- suffers from lack of sensitivity of the acetyl stored by addition of ferredoxin or methyl hydroxymate test. A more sensitive spectro- viologen. A test-tube assay referred to as the photometric assay is desirable and might be elastic assay was based on this finding (20, 35), based on the ferredoxin-dependent pyridine and is outlined in Fig. 6A; acetyl phosphate nucleotide reductase systems described below. production from pyruvate was proportional to ferredoxin concentration as shown in Fig. 6B Distribution (20). The phosphoroclastic assay was successfully Ferredoxin has been isolated from several used by Buchanan, Lovenberg, and Rabinowitz anaerobic bacteria and photosynthetic organ- (5) for crystallization of ferredoxin from several isms (Table 2), and is most prevalent among clostridial species (Table 1) and for our earlier the clostridia but is not confined to this group purification procedures (20, 35). (5, 18, 20, 35). It is generally true that ferredoxin is found in hydrogen-evolving species, but C. Hydrogenase Assay acidiurici is an interesting exception to this rule. The hydrogenase procedure of Peck and Gest This organism possesses a high content of ferre- (27) was readily adapted as an assay method for doxin (5) but does not produce H2 as a fer- ferredoxin (39). Evolution of hydrogen from mentation product (1); extracts have very low aqueous solutions of sodium dithionite (hydro- levels of hydrogenase activity (unpublished sulfite) was found by Peck and Gest to be greatly data). Ferredoxin, though, is required for pyru- enhanced by the addition of methyl or benzyl vate oxidation in C. acidiurici and appears to viologen. Ferredoxin replaced methyl viologen serve an important role, as will be discussed in this assay, and conditions were defined whereby later (41). evolution from dithionite was proportional to A thermostable form of ferredoxin has been ferredoxin concentration (39). The hydrogenase isolated from C. thermosaccharolyticum, a thermo- assay is in general more tedious and is not used philic anaerobe with an optimal growth tempera- routinely. ture of 55 C (42). Ferredoxin from C. thermosac- VOL. 28, 1964 FERREDOXIN 503 TABLE 2. Ferredoxin from bacteria (5, 33, 35 to 42) of Desuifovibrio desulfuricans. Ferredoxin did not substitute for methyl viologen as an efficient Organism Ferredoxin electron carrier between hydrogenase adenosine- Clostridium pasteurianum ...... + 5-phosphosulfate (APS) reductase or for the C. butyricum ...... + H2-sulfite reductase system (40). Low-potential C. lactoacetophilum . . + cytochrome C3, described by Postgate (28), may C. acidiurici ...... + play an important role in the reduction of inor- C. tetanomorphum ...... + ganic sulfur compounds by these organisms. C. kluyveri ...... + Extracts of hydrogenomonads, Azotobacter spe- C. thermosaccharolyticum . . + cies, and other aerobic bacteria have not yielded C. cylindrosporum ...... + ferredoxin activity, although low-potential car- C. sporogenes ...... + riers must be required in these organisms. C. nigrificans. Micrococcus lactilyticus . . + PROPERTIES OF CRYSTALLINE FERREDOXIN Peptostreptococcus elsdenii LC . . + Diplococcus glycinophilus . . + Oxidized and Reduced States Methanobacter omelianskii . . + Butyribacterium rettgeri ...... + Reducing agents such as sodium hydrosulfite Rhodospirillum rubrum . . + (dithionite) chemically convert a dark-brown Chromatium ...... + form of ferredoxin to a "leuco" or colorless form. Desulfovibrio desulfuricans . . + The dark-brown form returns after shaking in Streptococcus allantoicus. the air, and the process may be repeated. The Lactobacillus delbrueckii. dark-brown form is the oxidized form, and the Pseudomonad (isolate). colorless form is a reduced state; it is believed Escherichia coli (aerobic, anaerobic). that the dark-brown form possesses a Aerobacter aerogenes. single Bacillus macerans. additional electron, as was demonstrated with spinach chloroplast ferredoxin (43). Interaction with oxygen or other electron-accepting sub- charolyticum was able to withstand elevated stances oxidizes ferredoxin, returning it to the temperatures better than ferredoxin from C. original dark-brown state. Physiologically, ferre- pasteurianum but otherwise exhibited similar doxin is reduced by a variety of substrates, spectral and catalytic properties. Isolation of including pyruvate and H2; these enzymatic ferredoxin from photosynthetic bacteria (Rhodo- reductions will be described in detail in a later spirillum rubrum and Chromatium) was described section. by Tagawa and Arnon (33). Using a spectral method, Whatley, Tagawa, Extracts made from other important fermenta- and Arnon (43) compared the amount of chloro- tive organisms such as Escherichia coli and plast ferredoxin oxidized with the amount of Aerobacter aerogenes do not possess ferredoxin reduced NADP (NADPH) formed during the activity; this raises an important question enzymatic oxidation of chloroplast ferredoxin by regarding the mechanism of H2 formation by the NADP reductase. Their data indicated that 1 enteric group. Another low-potential carrier is mole of NADP was oxidized by 2 moles of probably utilized in place of ferredoxin; Gray et ferredoxin, according to the equation: al. (8) showed that a c-type cytochrome is syn- NADP reductame thesized during anaerobic growth by these 2 ferredoxin + NADP organisms, the development of hydrogenylase (reduced) and hydrogenase activity occurring simultane- NADPH + 2 ferredoxin ously with induction of the cytochrome (8). These (oxidized) experiments suggest that electrons from formate flow through low-potential cytochrome for H2 This experiment with spinach chloroplast ferre- evolution: doxin showed that the reduction and oxidation The role of ferredoxin in the sulfate-reducing of ferredoxin occurred by single electron transfer, species is not clear. Akagi (unpublished data) two reduced ferredoxin molecules being required found no detectable ferredoxin in C. nigrificans to reduce one molecule of NADP. Similarly, bac- extracts and only small quantities in most strains terial ferredoxin is believed to function by one 504 VALENTINE BACTERIOL. REV.

electron transport, but experimental evidence is TABLE 3. Amino acid composition of clostridial lacking on this point. ferredoxin (18) Spectra Source of ferredoxin Ferredoxin from five clostridial species has Amino acid Aa P. essentially the same catalytic and chemical properties, including a maximal light absorption at 390 m,4 as shown in Fig. 7 (5, 18). The 390-mA is essentially abolished when ferredoxin is Glycine ...... 4.0* 4.0 4.0 5.0 4.0 peak Methionine...... 0.0 0.0 0.0 0.4 reduced (33). Crystalline preparationsO0.0of ferre- Tryptophan ...... 0.0 0.0 0.0 0.0 0.1 doxin from C. pasteurianum, C. tetanomorphum, Histidine ...... 0.0 0.0 0.0 0.0 0.3 C. cylindrosporum, C. acidiurici, and C. butyricum Lysine ...... 0.9 0.1 0.5 0.2 1.5 exhibited distinct species-specific absorption in Arginine...... 0 0.9 0.0 0.0 0.2 the ultraviolet regions of 280 to 300 m~i (Fig. 7), Leucine ... 0.0 0.1 1.0 0.1 1.2 Tyrosine ...... 0.9 1.4 0.9 0.0 0.7 Phenylalanine ...... 1.0 0.1 1.0 2.0 1.8 320 400 480 560 640 M2 Cystine ...... 6.8 3.4 7.4 7.1 5.9 Alanine ...... 7.8 8.5 6.6 6.7 7.3 Valine ...... 5.3 5.1 4.0 5.1 4.6 Isoleucine ...... 4.3 4.0 4.3 2.9 4.5 Aspartic acid ...... 7.8 7.7 6.8 8.8 4.5 Threonine ...... 1 0.0 0.9 1.8 2.8 0.8 Serine ...... 4.1 2.6 3.4 2.7 3.8 Glutamic acid ...... 4.1 4.0 6.0 5.3 6.4 Proline ...... 3.0 3.7 2.9 2.9 3.2 * Amino acid residues per molecule. which indicates certain structural differences in ferredoxin from different bacterial species (18). _ ll Composition

-D The amino acid composition of crystalline 0n ferredoxin from C. pasteurianum, C. acidiurici, C. butyricum, C. tetanomorphum, and C. cylindrosporum is shown in Table 3 (18). The five clostridial ferredoxins closely resemble each other in amino acid composition (Table 3). All contain a single basic amino acid, with the exception of C. butyricum which contains no basic amino acids. A large number of acidic amino acid residues accounts for the overall acidic nature of clostridial ferredoxins. Ferredoxin from C. pasteurianum was found to have an isoelectric point of about 3.7 (18). None of the clostridial ferredoxins examined contains the amino acids histidine, methionine, or tryptophan; other 280 360 440 520 600 680 differences in amino acid composition are ap- Wavelength (me) parent in Table 3. For example, arginine occurs FIG. 7. Absorption spectra of clostridial ferre- only in the ferredoxin of C. acidiurici; on the doxin [Lovenberg, Buchanan, and Rabinowitz, (18)]. other hand, this ferredoxin is the only one exam- (1) Clostridium pasteurianum, (2) C. acidiurici, (3) ined that is free from phenylalanine (18). C. tetanomorphum, (4) C. butyricum, (5) C. cylin- Calculation of the molecular weight of C. drosporum. pasteurianum ferredoxin from the amino acid, VOL. 28, 1964 FERREDOXIN 505 iron, and organic sulfide composition gave a electron donors. It is interesting to note that value of 6,012, in good agreement with the value all compounds capable of reducing ferredoxin of 6,000 obtained by sedimentation equilibrium are potential precursors of molecular hydrogen data (18). (for example, pyruvate, a-ketoglutarate, hy- Crystalline ferredoxin contains approximately poxanthine, formate, and dithionite; see redox 5 molecules of "labile sulfide" (H2S released scale, Fig. 2). upon acidification) and 5 molecules of nonheme iron (Fe++) per each 6,000 molecular weight Phosphoroclastic System units (5, 18). Sulfide called labile sulfide is loosely As early as 1942, Koepsell and Johnson (12) bound to the protein, as evidenced by its dis- found that pyruvate served as a substrate for sociation as H2S at acidic or basic pH (5, 18). hydrogen production by use of extracts of C. The relationship of iron and sulfide is not yet butylicum: known, but these molecules may function at the pyruvate + phosphate catalytic center of ferredoxin. The binding of enzymes iron to ferredoxin was discussed recently by acetyl phosphate + C02 + H2 Lovenberg, Buchanan, and Rabinowitz (18) as follows: Koepsell and Johnson's experiment is easily "The iron of native ferredoxin is either repeated. A few milligrams of dried clostridial partially or entirely in the ferrous state as extract containing coenzyme A and other co- determined by the o-phenanthroline method. factors are added to a test tube containing Treatment of ferredoxin with the sulfhydryl phosphate buffer and pyruvate; within a few reagent, sodium mersalyl, causes a bleaching minutes, bubbles of gas resulting from pyruvate of the visible spectrum with the release of decomposition begin to form and rise along the iron and inorganic sulfide. The mersalyl- sides of the reaction tube. ferredoxin complex and apoferredoxin, i.e., Addition of dark-colored ferredoxin to the the protein free of iron and inorganic sulfide, above reaction tube results in immediate bleach- have been isolated and found to be devoid ing of ferredoxin to the leuco form; this property of enzymic activity in the C. pasteurianum allowed the direct reduction of ferredoxin to be clastic assay system. Under certain- condi- measured. About 50 to 60% of the ferredoxin pool tions, the ferredoxin-mersalyl complex, but appeared to be in the reduced form under steady- not apoferredoxin, can be reconstituted. state conditions during the elastic reaction This derivative is similar to the native according to the observed decrease in optical protein with respect to enzymic activity, density at 420 m1A (Fig. 8A) (21). The maximal absorption spectra, and iron and inorganic decrease in optical density obtainable (0.16 sulfide content. Iron59 is optical density units per 150 units of ferredoxin incorporated into ml in the mersalyl-ferredoxin complex, but not into per 3 Fig. 8A) was estimated by admitting the native protein. These observations are carbon monoxide, which inhibited the strong consistent with the existence of a 'covalent' hydrogenase reaction (21). Carbon monoxide bond between ferrous iron and sulfhydryl (CO) does not inhibit the activity of ferredoxin of cysteine." or pyruvic dehydrogenase, as indicated by the Iron and labile sulfide have been found associated rapid reduction of ferredoxin in an atmosphere of with spinach ferredoxin preparations, indicating CO (21). Reduction of ferredoxin by pyruvate the occurrence of iron with the clastic system is dependent upon co- general and sulfide in A ferredoxin from different sources (6, 11). Ferre- enzyme (Fig. 8B). In preparations from which doxin appears to be an interesting new type of coenzyme A had been removed, no detectable molecule-the chemistry and biosynthesis of reduction of ferredoxin by pyruvate occurred, ferredoxin are an open area for future research. until coenzyme A was added (Fig. 8B) (21). These results are in accord with the idea that ENZYMATIC REDUCTION OF FERREDOXIN coenzyme A is required for both the formation of acetyl phosphate and the reduction of ferre- In this section, I will discuss the mechanism doxin. Coenzyme A and ferredoxin appear to be of the enzymatic reduction of ferredoxin to its functioning together in enabling pyruvic dehy- leuco or colorless form by pyruvate and other drogenase to act. 506 VALENTINE BACTERIOL. REV.

A proposed mechanism for the pyruvate clastic that the limiting reaction in pyruvate oxidation system of C. pasteurianum is shown in Fig. 9. was concerned with electron transport; i.e., the The enzyme system appears to be composed of concentration of ferredoxin determined the rate pyruvic dehydrogenase, hydrogenase, and phos- of pyruvate oxidation in these organisms. In photransacetylase, together with coenzyme A later sections, additional references will be made and thiamine pyrophosphate. Ferredoxin has the to the clastic system because of its important important role of electron mediator between link to the reduced pyridine nucleotide reservoir pyruvic dehydrogenase and hydrogenase for H2 and N2 fixation. evolution. Recent interest in the clastic system has been focused on the close linkage of this TABLE 4. Role of ferredoxin in pyruvate oxidation by different bacteria*

B CA ,o DEAE cellu- Pyruvie+bFd(ox) Fd(red) Pyruvote+Fd(ox)- Fd(red) lose-treated , 0.16 Corbon monoxide 0.2225 Crude extract T extract _02 (no E Organism '7 0.122 carrier Ferre- 0 No 0.15 added) carrier doxin 'a T 0.08 added added

0 E 0.07 0.04 '0 60 units Clostridium pasteurianum...... 4.1 0.2 8.1 CoS odded no CoA C. lactoacetophilum ...... 2.2 0.1 5.0

0 2 3 4 5 10 11v O 5 C. acidiuricit ...... 1.7 0.1 2.4 Time (mir C. thermosaccharolyticum ...... 0..2 0.04 0.5 FIG. 8. (A) Enzymatic reduction of ferredoxin C. sporogenest ...... 0.8 0.6 0.6 with pyruvate by use of carbon monoxide to inhibit C. nigrificans ...... 0.4 0.4 4.1 hydrogenase (21). (B) Coenzyme A requirement for Micrococcus lactilyticus ...... 2.1 0.1 4.3 ferredoxin reduction from pyruvate (21). Peptostreptococcus elsdenii ...... 1.8 0.2 4.5 Butyribacterium rettgeri...... 0.1 0.0 2.7 Diplococcus glycinophilus ...... 1.1 0.1 1.4 Streptococcus allantoicus ...... 0.0 0.0 0.0 Rhodospirillum rubrum...... 0.0 0.0 0.0 CH3C-COOH FerredoxinA * Results expressed as micromoles of acetyl phosphate formed per milligram of protein per hour. For experimental conditions see refer- CZSoA ence 40. FIG. 9. Ferredoxin-linked pyruvate cleavage t NAD as final electron acceptor. (clastic reaction). : Glycine as final electron acceptor. reaction with N2 fixation, as shown in Fig. 9 a-Ketoglutarate (21, 22). Reduction of ferredoxin by another important A survey of pyruvate clastic reactions in several a-keto acid, a-ketoglutarate, has been observed anaerobic bacteria indicates that ferredoxin- in extracts of M. lactilyticus (40): linked pyruvate cleavage may occur generally ferredoxin (40) (Table 4). Addition of ferredoxin to extracts ca-ketoglutarate 3 of C. pasteurianum, M. lactilyticus (Veillonella H2 + 2CO2 + propionyl phosphate alcalescens), Peptostreptococcus elsdenii, C. lactoacetophilum, C. acidiurici, C. thermosaccharolyti- The pathway of H2 evolution from a-ketogluta- cum, Desulfovibrio desulfuricans, Butyribacterium rate has not been elucidated, but it appears to rettgeri, and Diplococcus glycinophilus showed involve a mechanism similar to that for pyruvate. marked stimulation of pyruvate oxidation (Table H2 evolution shows a complete dependence for 4). As shown previously, the addition of ferre- ferredoxin (40). These findings establish the doxin to crude extracts even before removal of a-ketoglutarate dehydrogenase system as another ferredoxin resulted in several-fold stimulation of source for strongly reducing electrons linked pyruvate oxidation (40). These results indicate with ferredoxin. VOL. 28, 1964 FERREDOXIN 507

Dithionite as H2 Precursor xanthine oxidation by a related organism, C. While not physiologically important, the cylindrosporum. Xanthine dehydrogenase (hypo- dithionite-H2 reaction has contributed to our xanthine dehydrogenase) was partially purified understanding of ferredoxin action. Crude hydro- from this source and was shown to contain, like genase preparations of C. pasteurianum catalyze the enzyme from milk and mammalian liver, the evolution of H2 from aqueous solutions of flavine adenine dinucleotide (FAD), nonheme sodium dithionite (hydrosulfite) (39): iron, and molybdenum (4). The in vivo function of this enzyme appears to be the reduction of chemical is not known whether cells adapted dithionite + ferredoxin reaction ferredoxin uric acid. It (oxidized) (reduced) to grow on hypoxanthine produce a different enzyme. hhydrogenase ferredoxin H2 + ferredoxin (reduced) (oxidized) Coupling With Formate Many organisms rapidly decompose formate, The rate of H2 evolution by this reaction was yielding H2 and (7): much slower than that observed by Peck and CO2 Gest (27). However, the rate of H2 production HCOOH -- H2 + CO2 Components of this system are formic dehydro-

Hypoxunthine Xonthine Ferredoxin genase and hydrogenase plus an unidentified oxidose (reduced) hydrogenose low-potential carrier (7). Interesting experiments H2 by Gray et al. (8), with the E. coli-A. aerogenes Xanthine Xonthine Ferredoxin system, indicate that a low-potential cytochrome oxidose (oxidized) may as a in (reduced) function carrier the formic hydro- genlyase reaction. Recent experiments with an FIG. 10. Mechanism of the hypoxanthine-H2 re- active formic dehydrogenase preparation from action of Micrococcus lactilyticus (35). C. acidiurici indicate that this enzyme couples with ferredoxin for pyridine nucleotide reduction from dithionite was found to be approximately (Fig. 16D), although in previous experiments equal to that from pyruvate. It is now clear that we were unable to demonstrate this reaction (40). both systems share ferredoxin as a common OF REDUCED electron carrier. OXIDATION FERREDOXIN Reduced ferredoxin is oxidized by a wide Hypoxanthine variety of electron acceptors, such as 02, nitrite, One of the simplest reactions yielding molecu- urate, hydroxylamine, and pyridine nucleotides. lar hydrogen as an end product is the oxidation With the exception of 02, the acceptors require of hypoxanthine to hydrogen and xanthine by specific enzymes (reductases); for example, re- M. lactilyticus (Fig. 10) (35, 44). Xanthine duced ferredoxin functions in conjunction with oxidase and hydrogenase were found to be urate reductase for conversion of urate to xan- constituents of this system, and the unidentified thine. The mechanism of these ferredoxin-linked electron carrier postulated by Whiteley and reductions will be discussed in this section. Ordal (44) was identified as ferredoxin (35). The entire hypoxanthine-H2 system appears to Urate be reversible; i.e., xanthine in an atmosphere of Whiteley and Douglas (44) first observed the H2 is slowly converted to hypoxanthine and H2-urate reductase system of M. lactilyticus and utilization of H2 is observed (44). were able to resolve the reaction into three com- A similar mechanism for hypoxanthine oxida- ponents including hydrogenase, urate reductase, tion is utilized by the purine-degrading organism and a low-potential carrier replaced by methyl C. acidiurici. In this organism, hypoxanthine viologen. Upon purification of these components, oxidation is coupled with reduction of pyridine the activity of the overall Hrurate reaction was nucleotides but does not give rise to hydrogen lost; this finding led Whiteley and Douglas (44) (W. Brill, unpublished data). Bradshaw and to postulate the existence of a low-potential Barker (4) investigated the mechanism of hypo- carrier which functioned between hydrogenase 508 VALENTINE BACTERIOL. REV. and urate reductase. In agreement with this The physiological role of these reactions in clos- postulation, ferredoxin was isolated from M. tridia is not known, but may be associated with lactilyticus and was found to serve as the "natu- N2 fixation by these organisms. Ferredoxin is ral" carrier for the H2-urate system as follows required as shown in Fig. 11, and the mechanism (35): for nitrite reduction shown in Fig. 12 has been proposed (38). Certain light-dependent reductions hydrogenaseh H2 + ferredoxin ferredoxin of nitrite and hydroxylamine by photosynthetic (oxidized) (reduced) tissues seem likely to have closely analogous ferredoxin + xanthine oxidase -> (reduced) 3H,2 + N02 - Fd )-- N H 3 reduced xanthine oxidase + ferredoxin (oxidized) reduced xanthine oxidase + urate -> xanthine oxidase + xanthine

TABLE 5. Pyruvate oxidation coupled with urate

reduction in Clostridium acidiurici: ._ ferredoxin requirement* , 220 Acetyl I Component omitted formedphosphatefrom pyruvate

umoles None ...... 2.7 Ferredoxin (0.18 mg) ...... 0.1 Urate (10 Mmoles) ...... 0.2 Pyruvate (50 /Amoles) ...... 0.0 0 5 10 15 Extract (8.6 mg, ferredoxin-free) .. 0.0 Tim '(minutes) * For experimental conditions see Table 1, FIG. 11. Ferredoxin requirement for nitrite re- reference 41. Urate replaced NAD as electron duction (40). acceptor for pyruvate oxidation. A similar system for urate Ferredoxin nitrite NH reduction is found in (oxidized) reductaseN 3 the purine-fermenting clostridia. With extracts hydrogenase of C. acidiurici, urate serves as electron acceptor H for pyruvate oxidation, coupling with ferredoxin Ferredoxin NO through pyruvic dehydrogenase (Table 5): (reduced) pyruv-ic pyruvate pyui FIG. 12. Proposed scheme for nitrite reduction by dehydrogenase the clostridia. urate + xanthine ferredoxinr reductase urate mechanisms. In this regard, Huzisige and Satoh The ferredoxin requirement for the pyruvate- (10) reported the isolation of photosynthetic urate system of C. acidiurici is shown in Table 5. nitrite reductase from spinach and discussed its The role of this reaction in C. acidiurici appears similarity with PPNR (spinach ferredoxin). to be for generation of xanthine, the first inter- Losada et al. (17) recently showed that both the mediate of urate decomposition. light-dependent and "dark" reductions of nitrite and hydroxylamine to ammonia by spinach Nitrite and Hydroxylamine require ferredoxin. These workers suggested that Nitrite and hydroxylamine are readily reduced nitrite reductase is a soluble flavoprotein (17). to ammonia by extracts of C. pasteurianum with No direct evidence is available for the existence molecular hydrogen as the reductant (37, 38, 40). of a specific bacterial ferredoxin-linked nitrite or VOL. 28, 1964 FERREDOXIN 509 hydroxylamine reductase (Fig. 11), though it many of these reductions and may replace the appears that ferredoxin does not react chemically unknown electron carrier (X) in Fig. 13. with these substrates (40). It was interesting to find that ferredoxin from spinach was an effective FERREDOXIN-LINKED PYRIDINE NUCLEOTIDE substitute for bacterial ferredoxin in the clos- REDUCTIONS tridial nitrite and hydroxylamine reductase Korices Factors systems (37). About 10 years ago, Seymour Korkes, working Reduction of C02 and Carbon Compounds in Earl Stadtman's laboratory, observed that nicotinamide adenine dinucleotide (NAD) or Several anaerobic bacteria can synthesize NADP reduction from H2 with extracts of C. acetate and methane from CO2, the metabolic kluyveri required unknown factors in addition hydrogen required for this reductive process being to hydrogenase (13, 14): NADPH Korkes CO2? Methane H, - * or cofactors NADHN rC02 B Acetate Because "boiling" did not completely destroy this activity (active preparations of ferredoxin H2 or (H) - [XH] - B from C. pasteurianum have been obtained by Butyryl P --- Butonol heating thick cell suspensions), Korkes factors Acetaldehyde C- Ethanol were designated as heat-stable cofactors in line Acetyl - P - Butyrate with concepts of the time. We now know that one of Korkes factors was most certainly ferre-

D doxin. Studies with the H2-pyridine nucleotide Glycine Acetate reduction system of C. kluyveri allowed Korkes to generalize further between hydrogen gas FIG. 13. Proposed scheme involving low-potential utilization by bacteria and photosynthetic carrier (X) for reduction of C02 and other carbon electron transport. He visualized a sequence of compounds. (A) Methanobacter omelianski (1, 47); events as follows (B) Clostridium thermaceticum (47), C. aceticum (14): (47), Diplococcus glycinophilus (2, 47); (C) C. hv kluyveri (31, 32); (D) C. sporogenes (25). H20 (A) XH2 ( ) NADH derived from degradation of substrates such as sugars and in certain cases from molecular H2 (B) hydrogen (1, 15, 47). Other anaerobes possess electron-transport chains leading from hydrogen Reaction A represents the chlorophyll-catalyzed gas to amino acids and fatty acids (2, 7, 20, 31, transfer of hydrogen from water to hydrogen 32). The electron-transport sequences involved acceptor, X, common to both pathways. Reaction in these reactions in most cases are poorly under- B represents the action of hydrogenase, and stood and for this reason will be described only reaction C is a common path operative in hy- briefly. The question of interest here is the pos- drogen-utilizing bacteria and in green plants for sible role of ferredoxin in these reactions-is the reduction of pyridine nucleotides (14). This ferredoxin a cofactor for reduction of CO2 and early scheme proposed by Korkes showed con- other carbon compounds? A series of reactions siderable insight into the problem of low-potential involving reduction of CO2 or other carbon electron transport and served as starting point for our present studies on pyridine nucleotide compounds is shown in Fig. 13. Organisms carry- reduction by the clostridia. ing out these reductions are given in the legend. The importance of the electron-transport chain H2-NADP connecting these reductive processes with the Cell-free extracts of C. pasteurianum and M. pool of metabolic hydrogen is apparent; it seems lactilyticus catalyze the rapid reduction of NADP likely that ferredoxin may play a key role in but not of NAD with molecular hydrogen (Fig. 510 VALENTINE BACTERIOL. REV. 14) (36, 45). This ferredoxin-dependent reaction, substituted for ferredoxin in the clostridial sys- probably similar to the one originally observed tem, but the specificity for NADP reduction was by Korkes, may be of general importance for lost, NAD now being reduced rapidly (36). This the clostridia and other bacteria where molecular finding indicates that the artificial dyes may hydrogen is available as reductant; for example, react chemically or through flavines or flavo- symbiotic conditions of the rumen may provide proteins, giving rise to nonspecific reductions of a suitable environment for both production and the pyridine nucleotides. utilization of hydrogen gas by the rumen bacteria (9). During periods of electron deficiency, the Pyruvate-NADP clostridia and other bacteria may utilize the H2-NADP reaction for pyridine nucleotide reduc- Pyruvate oxidation in C. pasteurianum is tion. In this manner, a balance between the directly coupled with the pyridine nucleotide hydrogen-producing and hydrogen-utilizing systems is maintained. The mechanism shown in H2 - Fd --TPN Fig. 14 for the H2-NADP reaction of C. pasteurianum appears to be closely analogous with the electron-transport sequence leading to NADPH 160k hydrogenose TPN reductose

H2 = > Ferredoxin EZ z> TPN a) N 120H FIG. 14. Mechanism of the H2-nicotinamide adenine dinucleotide phosphate reaction. cI It 80O- generation by green plants (33). In this sense, molecular hydrogen possesses reducing power 40 equivalent to that of the light-activated electrons minus Ferredoxin formed during photosynthesis (33). / --/o--o- O-o In the clostridial system, H2 is activated by ..1' 01 ooo- hydrogenase, and the electrons are transferred 0 20 40 60 80 to ferredoxin. Ferredoxin mediates the reduction Time (minutes) of NADP in conjunction with NADP reductase. It is interesting to note that Shin, Tagawa, and FIG. 15. Hydrogen-nicotinamide adenine di- Arnon (30) recently crystallized spinach NADP nucleotide phosphate reaction of Clostridium pas- ferredoxin as cofactor (36). reductase which catalyzes the ferredoxin-medi- teurianum: of similar have ated reduction NADP; enzymes NADP been implicated in the clostridial system for system through the ferredoxin-linked reduction of NADP (NADP reductase) and reductase system (36): NAD (NAD reductase) (36, 41). The ferredoxin pyruvate y requirement for the H2-NADP system of C. dehydrogenase

pasteurianum is shown in Fig. 15; a similar reac- TPN,T TPNH ferredoxin TPN tion was described by Tagawa and Arnon (33). reductase TPN These authors demonstrated the reduction of NADP in the dark with a system composed of Again, NADP but not NAD serves as electron bacterial ferredoxin, crude clostridial hydro- acceptor for pyruvate oxidation, NADP reduction genase, and a flavoprotein fraction (NADP from pyruvate being dependent on ferredoxin reductase) from spinach (33). In the present (Fig. 16A). The pyruvate-NADP reaction is experiments, it was found unnecessary to add the probably of great importance for C. pasteurianum, spinach flavoprotein, since sufficient quantities supplying reduced NADP for cellular synthesis. of the reductase fraction were already present This reaction also illustrates the importance of in the crude clostridial hydrogenase preparation ferredoxin as a coupling agent for electron flow (36). The viologen dyes (methyl and benzyl) from keto acid-level oxidations to the pyridine VOL. 28, 1964 FERREDOXIN 511 nucleotide pool, and may be generally used by of the C. acidiurici system are shown in Fig. 16B anaerobic bacteria. and 16C. Pyruvate again appears to be one of the major sources of reducing power for cellular Ferredoxin-NAD System synthesis in this organism, and it is interesting An interesting electron-transport chain has that none of the electrons from pyruvate are recently been elucidated in the urate-fermenting released as H2 (hydrogenase is absent). Figure 18 bacterium C. acidiurmi (41). Pyruvate oxidation illustrates the important role played by ferre- in this organism is coupled with pyridine nucleo- doxin in the electron-transport chain of C. acidi-

Pyruvate Ferredoxin DPNH TPN 0.4s0 Pyruvote Fd-TPN Pyruvate-Fd-DPN (oxidized) 0.20 -:A B Ferredoxin 0.3s0 - Ferredoxin added (TPN) added C02,ocetyl- P Ferredoxin DPN TPNH 0.2!O ad (reduced) 0.10) Pyruvic DPN Trans- 1r Ferredoxin dehydrogenase reductose hydrogenase added (DPN) 10 a, minus Ferredoxir minus Ferredoxin FIG. 17. Mechanism of nicotinamide adenine 0.0 0.C 0 3 6 0 0.5 1.0 dinucleotide reduction from pyruvate by Clostridium I z Pyruvote-'Fd- DPN -T P N Formate-.Fd -D PN acidiurici (14).

0.40 -C DPN D C] (2.8 0 z pumoles) 51

0.30 Ferredoxin added Hypoxonthine 0D DPN TPN (2.6/Lmoles) a/ 0.20 - + DPN(O.028 025 TPN 0 /0 i TPN(2.6)umoles) minus Ferredain Formote Ferredoxin 0.00 0.5 ° 0 .0 1.5 0 5 0 Time (minutes) Methylene -teira- FIG. 16. (A) Ferredoxin-linked pyruvate-nicotin- Pyruv0te hydrofolic acid amide adenine dinucleotide phosphate system of Pyruvate Clostridium pasteurianum (36). (B) Cofactor role of ferredoxin for the pyruvate-nicotinamide adenine dinucleotide system of C. acidiurici (41). (C) Fer- FIG. 18. Ferredoxin-linked electron-transport sys- redoxin-nicotinamide adenine dinucleotide system: tem of Clostridium acidiurici. (1) Hypoxanthine pyruvate oxidation by C. acidiurici linked specifi- dehydrogenase, (2) formic dehydrogenase, (3) cally with nicotinamide adenine dinucleotide (41). pyruvic dehydrogenase, (4) nicotinamide adenine (D) Formate oxidation by C. acidiurici coupled with dinucleotide reductase, (5) pyridine nucleotide trans- nicotinamide adenine dinucleotide reduction: fer- hydrogenase, (6) urate reductase, (7) methylene- redoxin requirement (curve, courtesy, W. J. Brill, tetrahydrofolate reductase. unpublished data). Conditions similar to B and C with 100 Mmoles of sodium formate replacing pyru- urici. In addition to reduction of pyridine nucleo- vate. tides, ferredoxin mediates electrons for urate reduction to xanthine (the first step of urate tide reduction (Fig. 17), a key reaction being decomposition) by a mechanism similar to that ferredoxin-linked NAD reduction (Fig. 17). This described for M. lactilyticus (35). In the C. acidi- is the first example of a ferredoxin-NAD reac- urici reaction, urate serves as electron acceptor tion, and may serve as an important analogy for for pyruvate oxidation; this coupled oxidation- photo-NAD reduction by photosynthetic bac- reduction reaction results in the formation of the teria (3). Electrons from pyruvate flow first to important energy-yielding metabolite, acetyl ferredoxin, which mediates the reduction of NAD phosphate. Electrons from formate (Fig. 18) (Fig. 17). NADPH may be generated from also flow through ferredoxin and are coupled NADH by transhydrogenase action (Fig. 17). with pyridine nucleotide reduction. As indicated The ferredoxin requirement and NAD specificity by these findings, ferredoxin is used extensively 512 VALENTINE BACTERIOL. REV. as an oxidation-reduction catalyst during urate for the nitrogenase system as shown in Fig. 20. decomposition, and plays a central role as elec- In Fig. 20, X1 and X2 represent hypothetical in- tron mediator coupling together oxidative and termediates formed during reduction of molec- reductive processes (Fig. 18). For further details ular nitrogen to ammonia by the hypothetical of the mechanism of urate fermentation by C. N2-reductase; X1 and X2 probably remain closely acidiurici and related organisms, see the review bound to the enzyme surface where they may be by Barker (2). converted to ammonia. Whether or not ferredoxin is the immediate electron donor for the "6 elec- FERREDOXIN AND BIOLOGICAL tron" reduction of N2 to ammonia by N2 reduc- NITROGEN FIxATION tase has not been determined. While other elec- The low redox potential of ferredoxin makes tron transport coenzymes may be required for it an ideal carrier or reductant for conversion certain reductive steps, it is possible that ferre-

0 Crude extract ai) N2 Cl) 15 0 Un xl (60 Ferredoxin jj a) -o X2 101 cm ._0N .(na1) FE;NH cU FIG. 20. Hypothetical scheme for N2 fixation involving ferredoxin and N2 reductase. ZI rO 5 z doxin mediates the complete reduction of N2 to 25 ml cell extract treate with 750 mg DEAE cellu lose ammonia. It is also interesting that the mech- / ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Iase anism of the ferredoxin-linked nitrogenase system 0 50 100 150 200 of C. pasteurianum appears to follow closely the FD (ag) general pattern for ferredoxin-coupled transport terminating in important reductive reactions-a FIG. 19. Ferredoxin requirement1 of fixation (21). for nitrogen pattern established through studies several of the reactions described above. The presence of f rrodnrin in mn.nv 0nnnprnhie nrwqni'qfqmc n.- ammonia in the Of N2 to nitrogen-fixing organ- ble of N2-fixation may indicate its general role isms; its widespread use as a reductant and its for reduction these species. requirement in pyruvate oxidation by extracts nitrogen by of C. pasteurianum capable of reducing molecular COENZYME NATURE OF FERREDOXIN nitrogen support this view (21). Coenzyme for Reductions and Oxidations Ferredoxin Requirements for Nitrogen Fixation Ferredoxin-linked reactions display specificity in regard to electron flow to and from ferredoxin. Mortenson (22) recently demonstrated the Thus, reduced ferredoxin works in conjunction ferredoxin requirements for N2-fixation by ex- with specific reductases and usually does not of of tracts C. pasteurianum by use extracts freed with substrates such as from ferredoxin by DEAE-cellulose treatment interact chemically NAD, (Fig. 19). He has shown that the pyruvic dehydro- NADP, or urate; in spite of the strongly favor- genase of this organism generates reduced ferre- able potential, incubation of reduced ferredoxin doxin which serves as a source of electrons for with urate, NAD, or NADP does not result in the conversion of molecular nitrogen to ammonia. reduced products. Instead, the reducing complex Ferredoxin may function as an electron carrier is composed of reduced ferredoxin plus the neces- VOL. 28, 1964 FERREDOXIN 513

A..

_ /.. .ifillmo- .

NW'

.1 i

-.R .;r

FIG. 21. Photomicrograph of crystals of Chromatiumferredoxin (kindly supplied by R. Bachofen, Y. Oda, and D. I. Arnon). sary reductase, for example, urate reductase or C. pasteurianum, ferredoxin interacted specifi- pyridine nucleotide reductase. cally with NADP, no NAD being reduced under An interesting example of the high degree of any of the experimental conditions tested (36). specificity displayed by ferredoxin-linked reduc- Because of the absence of pyridine nucleotide tases is illustrated by the pyridine nucleotide transhydrogenase, NAD was not reduced even reductases from different species of clostridia. In when small amounts of NADP were added to 514 VALENTINE BACTERIOL. REV. spark the reaction. On the other hand, C. acidi- production, photophosphorylation, and photo- urici extracts catalyzed a rapid ferredoxin-linked chemical nitrogen fixation, it appears likely that reduction of NAD but not NADP (41). These ferredoxin may play a role in these reactions. experiments suggested the presence of NADP- and NAD-specific reductases in these organisms, Comparison with Clostridia although these enzymes have not been isolated. An important experiment showing the equiva- In one sense, ferredoxin may be regarded as an lence of bacterial and spinach ferredoxin was electron-transport coenzyme much in the same performed by Tagawa and Arnon (33). Paneque manner as pyridine nucleotides are considered and Arnon (26) found that a small amount of oxidation-reduction coenzymes. crude C. pasteurianum hydrogenase was required

TABLE 6. Activity of ferredoxin

Reaction

Source z C Reference

C. thermosaccharolyticum. + + 42 Micrococcus lactilyticus...... ++ + + + 35 Chromatium...... >.+ + 33 Rhodospirillum rurum ...... + + 33 Spinach...... + + + + + + 33, 37

PHOTOSYNTHETIc FERREDOXIN E 12) Chromatium hy > Cr- Ferredoxin H2 Ferredoxin was first isolated from the photosynthetic bacterium, Chromatium, under the FIG. 22. Photoproduction of molecular hydrogen, and clostridial name of triphosphopyridine nucleotide reductase with plant chloroplasts, ferredoxin, hydrogenase (26). (TPNR; 16; see Fig. 21 for pictures of crystals of Chromatium ferredoxin, kindly furnished by R. Bachofen, Y. Oda, and D. I. Arnon). When for the photoproduction of hydrogen gas by The cell- Chromatium ferredoxin (TPNR) was tested by ferredoxin-free chloroplasts (Fig. 22). addition to washed chloroplasts deprived of their free extract of C. pasteurianum contained ferredoxin which about of own ferredoxin (also called photosynthetic pyri- brought photoproduction dine nucleotide reductase) the bacterial ferre- hydrogen gas by linking the photochemical ap- of with the added bac- doxin was similar to chloroplast ferredoxin paratus the chloroplast (photosynthetic pyridine nucleotide reductase) terial hydrogenase; clostridial ferredoxin was able in stimulating the photoreduction of NADP; to accept the light-activated electrons emitted the events and transfer NADP was preferentially reduced over NAD during photochemical (16). These experiments establish the catalytic them to hydrogenase (Fig. 22) (33). activity of ferredoxin from Chromatium when Recent work has indicated that photosynthetic added to the chloroplast system, but similar ferredoxin is functionally interchangeable with stimulation of electron-transport reactions in clostridial ferredoxin for most reactions tested; Chromatium has not been reported. Although no for example, spinach ferredoxin serves as electron experimental evidence is available which dem- carrier for several ferredoxin-dependent oxida- onstrates ferredoxin as a carrier in such interest- tion-reduction reactions in clostridia (37). Like- ing bacterial reactions as photochemical hydrogen wise, clostridial ferredoxin completely replaces VOL. 28, 1964 FERREDOXIN 515 spinach ferredoxin in the chloroplast reactions 3. BASSHAM, J. A. 1963. Photosynthesis: energet- (33). A summary of the ferredoxin-dependent ics and related topics. Advan. Enzymol. reactions is shown in Table 6. As seen from Table 25:39-117. 6, ferredoxins from eight photosynthetic and 4. BRADSHAW, W. H., AND H. A. BARKER. 1960. Purification and properties of xanthine de- nonphotosynthetic organisms are almost all capa- hydrogenase from Clostridium cylindro- ble of functioning interchangeably both in the sporum. J. Biol. Chem. 235:3620-3629. light and in the dark. [For other interesting 5. BUCHANAN, B. B., W. LOVENBERG, AND J. C. experiments on the role of ferredoxins in photo- RABINOWITZ. 1963. A comparison of clostrid- synthesis and photoproduction of H2, the reader ial ferredoxins. Proc. Natl. Acad. Sci. is referred to articles by Tagawa, Whatley, Arnon, U.S. 49:345-353. and co-workers (33, 34).] 6. FRY, K. T., AND A. SAN PIETRO. 1962. Studies on photosynthetic pyridine nucleotide re- Ferredoxin Versus Photosynthetic Pyridine ductase. Biochem. Biophys. Res. Commun. Nucleotide Reductase 9:218-225. 7. GEST, H. 1954. Oxidation and evolution of As discussed by Tagawa and Arnon (33), the molecular hydrogen by microorganisms. name photosynthetic pyridine nucleotide reduc- Bacteriol. Rev. 18:43-73. tase appears to be inappropriate for the ferre- 8. GRAY, C. T., J. W. T. WIMPENNY, D. E. doxin-like compounds isolated recently from HUGHES, AND M. RANLETT. 1963. A soluble bacteria and photosynthetic tissue [see discus- C-type cytochrome from anaerobically sion by San Pietro (30)]. Photosynthetic pyridine grown E. coli and various enterobac- nucleotide reductase implies a close relationship teriaceae. Biochim. Biophys. Acta 67:157- 160. with photochemical events; it is clear that non- 9. HUNGATE, R. E. 1962. Symbiotic associations: photosynthetic bacteria utilize ferredoxins for the rumen bacteria, p. 266-297. In P. S. many important "dark" oxidation-reduction Nutman and B. Morse [ed.], Symbiotic reactions. In fact, all ferredoxin reactions are dark associations. Cambridge University Press, processes not directly associated with the light Cambridge. reactions. The term "reductase" implies that 10. HuzISIGE, H., AND K. SATOH. 1961. Photosyn- ferredoxin is an enzyme, and this is probably also thetic nitrite reductase. Botan. Mag. (To- inappropriate in view of the coenzyme nature of kyo) 74:178-185. ferredoxin discussed earlier. It is, therefore, pro- 11. KATOH, S., AND A. TAKAMIYA. 1963. The iron- class of carriers be protein binding in photosynthetic pyridine posed that this low-potential nucleotide reductase. Arch. Biochem. Bio- named simply ferredoxins in view of their close phys. 102:189-200. functional similarity and presence in many non- 12. KOEPSELL, J. H., AND M. J. JOHNSON. 1942. photosynthetic organisms. Dissimilation of pyruvic acid by cell-free preparations of Clostridium butylicum. J. ACKNOWLEDGMENTS Biol. Chem. 145:379-386. 13. KORKES, S.1952. Discussion, p. 502-503. In W. E. I would especially like to thank R. S. Wolfe in McElroy and B. Glass [ed.], Phosphorus me- whose laboratory much of the early work with tabolism. The Johns Hopkins Press, Balti- ferredoxin was carried out. I am also indebted to more. W. J. Brill for permission to use certain unpub- 14. KORKES, S. 1955. Enzymatic reduction of lished experiments. I am grateful to W. Loven- pyridine nucleotides by molecular hydrogen. berg, L. E. Mortenson, J. C. Rabinowitz, D. I. J. Biol. Chem. 216:737-748. Arnon, and J. M. Akagi for kindly sending pre- 15. KORNBERG, H. L., AND S. R. ELSDEN. 1961. prints and other materials for this review. The metabolism of 2-carbon compounds by microorganisms. Advan. Enzymol. 23:410- LITERATURE CITED 470. 16. LOSADA, M., F. R. WHATLEY, AND D. I. ARNON. 1. BARKER, H. A. 1956. Bacterial fermentations, 1961. Separation of two light reactions in p. 1-28. John Wiley & Sons, Inc., New York. noncyclic photo-phosphorylation of green 2. BARKER, H. A. 1961. Fermentations of nitrog- plants. 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ERRATUM PHYTOTOXIC SUBSTANCES FROM SOIL MICROORGANISMS AND CROP RESIDUES T. M. McCALLA AND F. A. HASKINS Soil and Water Conservation Research Division, U.S. Department of Agriculture, and Nebraska Agricultural Experiment Station, University of Nebraska, Lincoln, Nebraska Volume 28, no. 2, page 192, col. 2, line 21: change "substances inhibitory to plant growth" to "substances stimulative to plant growth."