Ferredoxin Concentration Research Articles

Biomimetic photosynthetic system: Shedding light on the restoration of cell metabolism

Biomimetic photosynthetic system: Shedding light on the restoration of cell metabolism

Electron flow of biological H2 production by sludge under simple thermal treatment: Kinetic study

Mixed culture sludge has been widely used as a microbial consortium for biohydrogen production. Simple thermal treatment of sludge is usually required in order to eliminate any H2-consuming bacteria that would reduce H2 production. In this study, thermal treatment of sludge was carried out at various temperatures. Electron flow model was then applied in order to assess community structure in the sludge upon thermal treatment for biohydrogen production. Results show that the dominant electron sink was acetate (150–217 eˉ meq/mol glucose). The electron equivalent (eˉ eq) balances were within 0.8–18% for all experiments. Treatment at 100 °C attained the highest H2 yield of 3.44 mol H2/mol glucose from the stoichiometric reaction. As the treatment temperature increased from 80 to 100 °C, the computed acetyl-CoA and reduced form of ferredoxin (Fdred) concentrations increased from 13.01 to 17.34 eˉ eq (1.63–2.17 mol) and 1.34 to 4.18 eˉ eq (0.67–2.09 mol), respectively. The NADH2 balance error varied from 3 to 10% and the term eˉ(Fd↔NADH2) (m) in the NADH2 balance was NADH2 consumption (m = −1). The H2 production was mainly via the Fd:hydrogenase system and this is supported with a good NADH2 balance. Using the modified Gompertz model, the highest maximum H2 production potential was 1194 mL whereas the maximum rate of H2 production was 357 mL/h recorded at 100 °C of treatment.

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

Electron bifurcation is a fundamental strategy of energy coupling originally discovered in the Q-cycle of many organisms. Recently a flavin-based electron bifurcation has been detected in anaerobes, first in clostridia and later in acetogens and methanogens. It enables anaerobic bacteria and archaea to reduce the low-potential [4Fe-4S] clusters of ferredoxin, which increases the efficiency of the substrate level and electron transport phosphorylations. Here we characterize the bifurcating electron transferring flavoprotein (EtfAf) and butyryl-CoA dehydrogenase (BcdAf) of Acidaminococcus fermentans, which couple the exergonic reduction of crotonyl-CoA to butyryl-CoA to the endergonic reduction of ferredoxin both with NADH. EtfAf contains one FAD (α-FAD) in subunit α and a second FAD (β-FAD) in subunit β. The distance between the two isoalloxazine rings is 18 Å. The EtfAf-NAD(+) complex structure revealed β-FAD as acceptor of the hydride of NADH. The formed β-FADH(-) is considered as the bifurcating electron donor. As a result of a domain movement, α-FAD is able to approach β-FADH(-) by about 4 Å and to take up one electron yielding a stable anionic semiquinone, α-FAD, which donates this electron further to Dh-FAD of BcdAf after a second domain movement. The remaining non-stabilized neutral semiquinone, β-FADH(•), immediately reduces ferredoxin. Repetition of this process affords a second reduced ferredoxin and Dh-FADH(-) that converts crotonyl-CoA to butyryl-CoA.

Accelerated electron transport from photosystem I to redox partners by covalently linked ferredoxin

Photosystem I is a highly efficient and potent light-induced reductase that is considered to be an appealing target for integration into hybrid solar fuel production systems. However, rapid transport of multiple electrons from the reducing end of photosystem I to downstream processes in vivo is limited by the diffusion of its native redox partner ferredoxin that is a single electron carrier. Here, we describe the design and construction of a faster electron transfer interface based on anchoring ferredoxin to the reducing end of photosystem I thereby confining the diffusion space of ferredoxin to the near vicinity of its photosystem I binding and reduction site. This was achieved by fusing ferredoxin to the PsaE subunit of photosystem I by a flexible peptide linker and reconstituting PSI in vitro with the new fusion protein. A computational algorithm was developed in order to determine the optimal linker length that will confine ferredoxin to the vicinity of photosystem I's reducing end without restricting the formation of electron transfer complexes. According to the calculation, we reconstituted photosystem I with three fusion proteins comprising PsaE and ferredoxin separated by linkers of different lengths, namely 14, 19, and 25 amino acids, and tested their effect on electron transfer rates from photosystem I to downstream processes. Indeed, we found a significant enhancement of light dependent NADPH synthesis using photosystems containing the PsaE-ferredoxin fusion proteins, equivalent to a ten-fold increase in soluble ferredoxin concentration. We propose that such a system could be used for other ferredoxin dependent redox reactions, such as the enzymatic production of hydrogen, a promising alternative fuel. As the system is comprised entirely of natural amino acids and biological cofactors, it could be integrated into the energy conversion apparatus of photosynthetic organisms by genetic engineering.

Detection of the Photosystem I:Ferredoxin Complex by Backscattering Interferometry

The dissociation constant K(d) of the photosystem I (PSI):ferredoxin complex has been measured by backscattering interferometry (BSI) with cyanobacterial PSI (350 kDa) and ferredoxin (10.5 kDa). The BSI signal, consisting of shifts for interference fringes resulting from a change in refractive index due to complex formation, was monitored as ferredoxin concentration was titrated. K(d) values of 0.14-0.38 microM were obtained with wild-type PSI whereas no complex was detectable with a PSI mutant containing a single mutation (R39Q) in the PsaE extrinsic subunit. These results are in quantitative agreement with previous functional determinations consisting in the detection of fast electron transfer within the complex. They provide evidence that the main contribution for the high affinity binding of ferredoxin to PSI is due to a single region of PsaE comprising arginine 39. They do not support the existence of a secondary binding site that could have escaped functional detection.

Ferredoxin and flavodoxin in eastern Antarctica pack ice

The presence, concentration and distribution of the iron regulated proteins, ferredoxin and flavodoxin, was investigated in pack ice off eastern Antarctica using SDS-PAGE gels. Bands corresponding to ferredoxin and/or flavodoxin were observed in all but eight of the 102 core sections analysed. Flavodoxin was found in most of the ice samples and was strongly correlated with chlorophyll a standing stock. The widespread distribution of flavodoxin here is not thought to indicate iron-limitation as many of the dominant species, such as Fragilariopsis cylindrus, Cylindrotheca closterium, are known to produce this protein under iron-replete conditions and thus the significant correlation between flavodoxin and biomass is likely to be the result of widespread constitutive flavodoxin expression among the diatoms that commonly inhabit sea ice. High concentrations of ferredoxin were predominantly derived from core sections on the floes closest to the continent and also in the upper portion of these floes. There was a consistent lack of ferredoxin expression in the high biomass bottom communities. The absence of ferredoxin is likely to indicate a reduced supply of iron but the significance of this reduced iron supply cannot be inferred on the basis of protein expression alone. Furthermore, in the present study the observed variability in the flavodoxin:ferredoxin ratio may not simply reflect the iron nutritional status of the community, but probably results from changes in the abundance of species capable of expressing ferredoxin.

Low levels of ferredoxin, ATP and leghemoglobin contribute to limited N2 fixation of peas (Pisum sativum L.) and alfalfa (Medicago sativa L.) under S deficiency conditions

Sulphur (S) has become a major limiting factor for plant production in industrial as well as in remote industrial rural areas. Limitation of S can reduce legume N2 fixation by affecting nodule development and function. In pot experiments with pea (Pisum sativum L.) and alfalfa (Medicago sativa L.), we investigated the influence of S on growth, ferredoxin, ATP and leghemoglobin concentrations. Addition of 200 mg S pot−1 increased yield of shoots, roots and nodules of both plant species significantly. However, the influence of S on nodule yield formation was most pronounced. Pea and alfalfa roots were found to have higher S concentrations than shoots and being up to 2.9 times the S concentration in the shoots of peas under S-sufficient conditions. Sulphur addition also increased N2 fixation significantly. The ferredoxin concentration in bacteroids of root nodules of pea was increased significantly by S only 10 weeks after planting and in bacteroids of root nodules of alfalfa 10 and 17 weeks after planting, while on per pot base the amounts of ferredoxin were higher throughout the experimental period of time. The ATP concentration of bacteroids of root nodules of both plant species as well as of mitochondria of root nodules of pea were significantly higher with optimum S supply. The effects of S deficiency on N2 fixation are likely to be caused by the shortage of ferredoxin and ATP. The amount of leghemoglobin was reduced in comparison to nodules of the S-sufficient plants.

Participation of ferredoxin in oxygen reduction by the photosynthetic electron transport chain

Oxygen reduction in a photosynthetic electron-transport chain (PETC) was studied in isolated pea thylakoids in the presence of either ferredoxin, or ferredoxin + NADP+, or cytochrome c. The contribution of the electron flow through ferredoxin to the total oxygen reduction was evaluated by comparing the rate of oxygen reduction and the rate of oxidation of reduced ferredoxin in the light. It was found that at ferredoxin concentrations optimal for NADP+ reduction, 30-50% of electrons transferred to oxygen went through ferredoxin both in the absence and presence of NADP+. However, the absolute rate of oxygen reduction by membrane components of PETC in the presence of NADP+ was 3-4 times less than that in the presence of ferredoxin alone and close to the rate of oxygen reduction in the presence of cytochrome c. It was assumed that a Photosystem I component, whose role in this process depends on the rate of electron outflow from terminal acceptors of this photosystem, participates in oxygen reduction, and this component is phylloquinone.

Electrochemical Study of a Reconstituted Photosynthetic Electron-Transfer Chain

A multi-enzyme electron-transfer chain involving solubilized photosystem I (PSI) as photocatalytic unit, cytochrome c6 and ferredoxin as electron carriers and ferredoxin/NADPH oxidoreductase (FNR) as electron acceptor was reconstituted in an electrochemical cell and studied by cyclic voltammetry. The working gold electrodes were modified to react selectively with cytochrome c6. Quantitative analysis of the photocatalytic current under continuous illumination allowed the determination of the values kon and koff for the ferredoxin/PSI interaction. An efficient recycling system for NADPH was established, and the dissociation constant of the oxidized ferredoxin/semiquinone FNR complex was extracted by modeling the catalytic efficiency of the chain as a function of ferredoxin concentration. The value determined hereby is consistent with a shift of -50 to -100 mV of the reduction potential of ferredoxin when complexed with FNR.

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans.

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from ( R)-2-hydroxyglutaryl-CoA to ( E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S](2+) cluster, 1.0 reduced riboflavin-5'-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S](1+/2+)-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg(2+)-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S](1+/2+)-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as -405 mV and -340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7-45 microM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 micromol/g protein at 7 microM Fe to 0.02 micromol/g at 45 microM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 micromol/g at 7-13 microM Fe to 1.1+/-0.1 micromol/g at 17-45 microM Fe.

Kinetics of the decomposition of [2Fe–2S] ferredoxin from spinach

Abstract The decomposition of spinach ferredoxin has been studied, at 37°C, in the presence of: buffer (pH 7.5); water; 0.01 M hydrochloric acid; and 0.01 M hydrochloric acid with pepsin enzyme (0.09, 0.19 and 0.38%). The hydrolysis of the protein appears to occur slowly in the presence of water alone, but is greatly accelerated by the addition of acid. This hydrolysis reaction is proposed to involve the denaturation of the protein chain to the extent that the [2Fe–2S] core is released from the protein. In the presence of 0.01 M HCl, total iron release occurs. Pepsin appears to then breakdown the peptide linkages within the apoprotein. UV–visible spectroscopy has demonstrated a two stage reaction with loss of the peak at 422 nm correlating with breakdown and loss of the [2Fe–2S] cluster, and loss of the peak at 277 nm demonstrating breakdown of the protein chain. Uniphasic kinetics were observed at 422 nm with the observed pseudo-first order rate constant having a linear dependence on ferredoxin concentration.

Ferredoxin reduction by photosystem I from Synechocystis sp. PCC 6803: toward an understanding of the respective roles of subunits PsaD and PsaE in ferredoxin binding.

The process of ferredoxin reduction by photosystem I has been extensively investigated by flash-absorption spectroscopy in psaD and psaE deleted mutants from Synechocystis sp. PCC 6803. In both mutants, the dissociation constant for the photosystem I/ferredoxin complex at pH 8 is considerably increased as compared to the wild type: approximately 25- and 100-fold increases are found for PsaD-less and PsaE-less photosystem I, respectively. However, at high ferredoxin concentrations, submicrosecond and microsecond kinetics of electron transfer similar to that observed in the wild type are present in both mutants. The presence of these fast kinetic components indicates that the relative positions of ferredoxin and of the terminal photosystem I acceptor are not significantly disturbed by the absence of either PsaD or PsaE. The second-order rate constant of ferredoxin reduction is lowered 10- and 2-fold for PsaD-less and PsaE-less photosystem I, respectively. Assuming a simple binding equilibrium between photosystem I and ferredoxin, PsaD appears to be important for the guiding of ferredoxin to its binding site (main effect on the association rate) whereas PsaE seems to control the photosystem I/ferredoxin complex lifetime (main effect on the dissociation rate). The properties of electron transfer from photosystem I to ferredoxin were also studied at pH 5. 8. In the psaE deleted mutant as in the wild type, the change of pH from 8 to 5.8 induces a 10-fold increase in affinity of ferredoxin for photosystem I. In the absence of PsaD, this pH effect is not observed, in favor of this subunit being mostly responsible for the low pH increased affinity.

In Vitro Cyclic Electron Transport in Barley Thylakoids follows Two Independent Pathways

In vitro cyclic electron transport around PSI was studied in thylakoids isolated from barley (Hordeum vulgare L.). Redox poising was obtained by using anaerobic conditions, preillumination, and the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Postillumination rates of P700+ re-reduction of 1 to 5 electrons s-1 were observed, depending on the conditions. The thylakoids supported two parallel paths of cyclic electron transport that were distinguishable by differences in antimycin sensitivity, saturation characteristics, and substrate specificity. The pathway most sensitive to antimycin was not saturated at ferredoxin concentrations up to 50 [mu]M, whereas the more insensitive pathway was saturated at 5 [mu]M ferredoxin. At the lower concentration of reduced ferredoxin, the antimycin-sensitive rate of P700+ re-reduction was lower than the antimycin-insensitive rate. The lower range of reduced ferredoxin concentrations are closer to in vivo conditions. Flavodoxin is shown to mediate cyclic electron transport. Flavodoxin was less efficient in mediating the antimycin-sensitive pathway but mediated the antimycin-insensitive pathway as efficiently as ferredoxin. Antibodies raised against ferredoxin:NADP+ oxidoreductase had no effect on either pathway for re-reduction of P700+. However, the ferredoxin: NADP+ oxidoreductase inhibitor 2[prime]-monophosphoadenosine-5[prime]-diphosphoribose was able to inhibit the antimycin-sensitive as well as the antimycin-insensitive pathway.

Ferrodoxin-dependent CO 2 fixation in bean sprouts

Extracts of bean sprouts are capable of reducing ferrodoxin and of catalyzing the incorporation of bicarbonate and acetyl coenzyme A into an organic compound that is likely to be pyruvate, in a reaction that requires reduced ferredoxin. The rate of the reaction, the first known for which ferredoxin appears to serve as the direct reductant for CO 2 fixation in a higher plant, depends on the concentrations of both ferredoxin and bicarbonate, with half-maximal rates being observed at ferredoxin and bicarbonate concentrations of 0.8 μM and 200 μM, respectively.

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.

Small subunits of Photosystem I reaction center complexes from Synechococcus elongatus. II. The psaE gene product has a role to promote interaction between the terminal electron acceptor and ferredoxin

Function of a subunit polypeptide (the psaE gene product) of Photosystem I (PS I) reaction center complexes was investigated by comparing the reactivity of the reduced iron-sulfur centers (F A/F B) − with ferredoxin among Synechococcus PS I complexes which had been variously depleted of this polypeptide. Ferredoxin at or below 1 μM can accept electrons from (F A/F B) − effectively competing with the back reaction between P-700 + and (F A/F B) − in the thylakoid membranes and PS I complexes that contained all the eight small subunits. The high reactivity of (F A/F B) − with low concentrations of ferredoxin was observed in PS I complexes which contain only the products of psaC, psaD and psaE genes but not in complexes which carry the psaC, psaD, psaL and psaK gene products but no psaE gene product. Varied amounts of the psaE gene product were extracted by treatment with different concentrations of a cationic detergent, dodecyltrimethylammonium bromide, and 2.5 M NaCl. The solubilized polypeptide was then reconstituted to the depleted complexes. The magnitudes of the back reaction that could be suppressed by addition of ferredoxin at or below 1 μM were well correlated to the amounts of the psaE polypeptide remained bound or rebound to the complexes. It is concluded that the product of the psaE gene has a role to promote the interaction between the terminal bound electron acceptor and ferredoxin. A high autooxidizability of (F A/F B) − and contrasting effects of lipophilic cations and anions on the rate of the back reaction from (F A/F B) − to P-700 + were also reported.

Solubilization and reconstitution of kidney 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases from vitamin D-replete pigs.

Pig kidney mitochondrial 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities have been solubilized with cholate/Emulgen 911 and reconstituted with NADPH, ferredoxin reductase and ferredoxin. All three of these components are required for full catalytic activity of both enzymes. Both products were identified by co-chromatography with authentic metabolites on both normal and reverse-phase h.p.l.c. using solvent systems which were shown to separate 10-oxo-19-nor-25-hydroxyvitamin D3 from 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3]. In addition, periodate treatment of the 24,25-(OH)2-D3 product resulted in complete loss of the product as measured by protein-binding assay. Further purification by p-chloroamphetamine-Sepharose chromatography of a solubilized extract from a pig fed a normal diet increased the specific content of the cytochrome P-450 from 0.019 to 0.239 nmol/mg and the 1 alpha-hydroxylase activity from 4.75 to 268 pmol/h per mg. Activity of the 24-hydroxylase in the crude solubilized extract was 6.3 pmol/h per mg, but was undetectable after partial purification by a p-chloroamphetamine-Sepharose column. However, further fractionation of this material by DEAE-Sepharose chromatography resulted in a further increase in 1 alpha-hydroxylase activity to 430 pmol/h per mg and detection of 24-hydroxylase in a separate fraction at a level of 53 pmol/h per mg. Production of 1,25-(OH)2-D3 was linear with time up to 2 h and was dependent upon ferredoxin concentration as well as cytochrome P-450 concentration in the range of 0-40 nM. In the presence of excess ferredoxin and adequate amounts of cytochrome P-450, 1,25-(OH)2-D3 production was also dependent upon substrate concentrations in the range of 0.25 to 2.5 microM yielding an estimated Km of 1 microM. In the presence of excess substrate and ferredoxin, the catalytic-centre activity of the enzyme was estimated to be 1 h-1.

Reduction of S-sulfoglutathione by cyanobacterial ferredoxin-sulfite reductase.

S-Sulfoglutathione is reduced by reduced ferredoxin and ferredoxin dependent sulfite reductase (Fd-SiR) from the cyanobacterium Spirulina platensis. The Km value calculated from a Lineweaver-Burk plot of the data was 6.0 × 10-5 m. A plot of ferredoxin concentration versus reduction activity of S-sulfoglutathione (GSSO3H) of Fd-SiR yielded a sigmoidal curve, giving a Hill coefficient (n) of 3.0. Antiserum against Fd-SiR strongly inhibits this reaction. In an experiment using a crude cell extract, the GSS03H reducing activity was only found at the site of Fd-SiR in gel filtration and immunoblotting.

Electron spin resonance studies of the ovary of the rat

Electron spin resonance spectra of rat ovaries, isolated ovarian compartments, and ovarian subcellular fractions were compared with spectra of rat adrenals. Rat ovaries were found to exhibit ESR signals similar to those previously described in studies of mammalian adrenal and testis. Observations were made at 113 K in an anaerobic environment. ESR signals of the low-spin ferric cytochrome P-450, the non-heme protein ferredoxin, and the non-heme glycoprotein transferrin were consistently observed in whole ovaries. The first two signals were detected in mitochondrial fractions isolated from ovaries, while only cytochrome P-450 was detected in microsomal fractions. Signals from ferredoxin and cytochrome P-450 were also consistently observed in both whole adrenals and adrenal mitochondrial fractions. However, in the microsomal fraction only cytochrome P-450 was present. The g values for the cytochrome P-450 and ferredoxin signals found in this study of ovaries were identical to those previously reported and also found in this study in spectra of rat adrenals. The concentration of ferredoxin per milligram wet mass in rat ovaries appears to be only one-sixth of that in the rat adrenal. The concentration of cytochrome P-450 appears to be only one-ninth of that in the adrenal. Signals from ferredoxin were detected in all ovarian compartments except granulosa cells isolated from Graafian follicles. The third signal, that of transferrin, while often observed in the spectra of whole ovaries, has been attributed to residual blood in the tissues examined. The effects of oxygen on these spectra has been found to be considerable and is discussed.

Ferredoxin and cytochrome P-450 scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization

The concentrations of cytochrome P-450 scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450 scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450 scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450 scc, was always adequate to saturate both the reductase and cytochrome P-450 scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450 scc and the concentration of ferredoxin minus ferredoxin reductase.