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Bacterial Ferredoxin' R 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-medi- ating 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 trans- port 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. acidi- urici, 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,
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