Interdomain communication revealed in the diabetes drug target mitoNEET

MitoNEET is a recently identified drug target for a commonly prescribed diabetes drug, Pioglitazone. It belongs to a previously uncharacterized ancient family of proteins for which the hallmark is the presence of a unique 39 amino acid CDGSH domain. In order to characterize the folding landscape of this novel fold, we performed thermodynamic simulations on MitoNEET using a structure-based model. Additionally, we implement a method of contact map clustering to partition out alternate pathways in folding. This cluster analysis reveals a detour late in folding and enables us to carefully examine the folding mechanism of each pathway rather than the macroscopic average. We observe that tightness in a region distal to the iron-sulfur cluster creates a constraint in folding and additionally appears to mediate communication in folding between the two domains of the protein. We demonstrate that by making changes at this site we are able to tweak the order of folding events in the cluster binding domain as well as decrease the barrier to folding.     

NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.     

Hypouricemia linked to an overproduction of nitric oxide is an early marker of oxidative stress in female subjects with type 1 diabetes

OBJECTIVE: The aim of this study is to verify whether, early in the course of type 1 diabetes and assuming hyperglycemia as the only risk factor, women demonstrate a change in oxidative status due to an interaction between nitric oxide (NO) and uric acid production. METHODS: Thirty-eight women with type 1 diabetes of less than 10 years' duration and with no diabetic complications were compared with 25 matched healthy female controls. Insulin, C-peptide, NO, HbA(1c) and oxidative stress metabolites were determined from venous blood samples taken from all patients after a 12 h overnight fast. Urine samples were used for urinary uric acid determination. RESULTS: Most oxidative stress metabolites were significantly increased (p < 0.0001), while plasmatic and urinary uric acid levels were significantly lower (p < 0.0001) in patients with type 1 diabetes compared with controls. Mean NO levels were inversely related to uricemia. Bivariate regression analysis showed a significant correlation between plasmatic uric acid and NO (p = 0.004), ascorbic acid (p = 0.042), triglycerides (p = 0.014) and HbA(1c) (p < 0.0001). Linear multivariate regression analysis showed a significant relationship between HbA(1c) and plasmatic uric acid (beta = - 0.465, p = 0.0004). CONCLUSIONS: Oxidative stress is already present in the early stages of type 1 diabetes. We conclude that the initial increase in oxidative stress could be linked to a reduction in plasmatic levels of uric acid, which is probably directly caused by an overproduction of NO.     

A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron–sulfur cluster that is necessary for activity. These findings provide an example of an iron–sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)–dependent amide bond formation in a natural product biosynthetic pathway.

Amide bond formation in natural product biosynthesis can be catalyzed by nonribosomal peptide synthetases (NRPSs) and ATP-grasp ligases including the ATP-dependent activation of amino acid substrates. The carboxyl group is activated by phosphorylation and adenylation, respectively, in ATP-grasp ligase and NRPS catalyzed reactions (1). Recently, a new family of enzymes has been found to catalyze amide bond formation using aminoacyl-transfer RNA (aa-tRNA) as an activated cosubstrate. aa-tRNA plays a profound role in cells connecting the messenger RNA (mRNA) and protein synthesis at the ribosome in primary metabolism (2). Interestingly, recent studies have revealed that aa-tRNA can also be involved in natural product biosynthesis (3). aa-tRNA–dependent enzymes involved in natural product biosynthesis mainly form three groups: amide bond-forming ligases homologous with FemX peptidyltransferases from cell wall biosynthesis (4), synthase enzymes including the cyclodipeptide synthase family (5, 6), and dehydratase enzymes in RiPP (ribosomally synthesized and posttranslationally modified peptide) synthesis (7).Blasticidin S (BS), a representative of the amino hexose pyrimidine nucleoside antibiotics, was first isolated from Streptomyces griseochromogenes (8). The structure of BS features a C2′, C3′-dehydrated pyrose ring that is connected with cytosine at C1′ and β-arginine at C4′ (9). BS shows a broad spectrum of biological activities and had been widely used as a fungicide to protect rice from blast diseases (10, 11). Presently, BS is commonly used as an efficient selection antibiotic for transformed mammalian cells that express appropriate resistance genes (12, 13).The structural features and commercial value of BS stimulated interest in its biosynthetic studies. Early feeding experiments and characterization of related metabolites determined that glucose, cytosine, L-arginine, and methionine are the basic precursors for BS biosynthesis (14). The biosynthesis gene cluster was reported in 2003 and enabled a proposal for the BS biosynthetic pathway (Fig. 1) (15). However, difficulty carrying out gene knockout experiments in the native producer, S. griseochromogenes, hindered the further characterization of each biosynthetic step. More recently, the BS biosynthetic cluster was engrafted into the chromosome of Streptomyces lividans to generate the genetically stable mutant strain WJ2, which is able to produce BS and thereby facilitating in vivo studies of the function of BS biosynthetic genes (13). Gene deletions in WJ2 determined the essential genes for BS biosynthesis including blsD-blsL that are transcribed in one direction and the divergently transcribed blsM. The first committed step of BS biosynthesis is the hydrolysis of cytidine monophosphate by BlsM to produce free cytosine, which is then condensed with UDP-glucuronic acid by BlsD to form cytosyl-glucuronic acid (CGA) (15, 16). The process from CGA to cytosinine, a putative intermediate for BS biosynthesis, remains to be determined. A radical SAM protein BlsE and an aminotransferase BlsH are possibly involved in this transformation (13, 15). The β-arginine moiety of BS is derived from L-arginine through a radical-mediated reaction catalyzed by BlsG, a 2,3-arginine aminomutase (15, 17). It remains unknown whether BlsI, a putative ligase, is involved in the formation of DBS (demethylblasticidin S) by coupling of the carboxyl group of β-arginine and the amido group at C4′ of cytosinine since mutant WJ2∆blsI only accumulates the very early intermediate CGA. It is worth noting that DBS cannot be directly methylated to BS by the N-methyltransferase BlsL, which was confirmed by in vitro biochemical characterization (18).Open in a separate windowFig. 1.The proposed biosynthetic pathway of BS. CMP: Cytidine 5-monophosphate; AdoMet: S-Adenosylmethionine.Addition of leucine to DBS at the β-amino group of the arginine side chain forms leucyldemethylblasticidin S (LDBS) which is then methylated by BlsL to form the penultimate product leucylblasticidin S (LBS) (18). Finally, PepN catalyzes the maturation of BS biosynthesis via the hydrolysis of the leucyl group of LBS (19). The cell toxicity of LDBS and LBS are much lower than DBS and BS. Therefore, it is proposed that the circuitous modifications of BS and DBS with a leucyl group function as the self-resistance mechanism during BS biosynthesis (16). Attempts to form LDBS from DBS in a S. griseochromogenes cell-free system were not successful. A plausible reason for the failure of LDBS formation was that even if LDBS could be formed, it would be readily hydrolyzed back to DBS by the conserved aminopeptidase PepN in the cell-free system (16, 19).The intermediacy of LBS and LDBS hints at the necessity of an extra ligase other than BlsI in BS biosynthesis (15). In this report, we disclose the mechanism of the cryptic leucylation involved in BS biosynthesis. By combining in vivo gene inactivation and in vitro biochemical assays, we demonstrate that BlsK catalyzes the tRNA-dependent transfer of a leucyl group to the DBS β-arginine moiety by directing leucyl-tRNA^Leu to the BS biosynthetic pathway. More interestingly, BlsK is determined to be an iron–sulfur enzyme that does not show similarity with any known iron–sulfur enzymes. BlsK contains a [3Fe-4S] cluster that is critical for its activity. An iron–sulfur protein was shown to be involved in the amide bond formation. These results pave the way to fully understand the self-resistance and biosynthesis of BS.     

蜕皮甾酮对2型糖尿病大鼠肾组织氧化应激的影响

目的探讨蜕皮甾酮对2型糖尿病(T2DM)大鼠肾组织氧化应激的影响。方法选择70只雄性SD大鼠,高脂喂养8周后,对60只用链脲佐菌素腹腔注射建立T2DM模型,然后分T2DM组、蜕皮甾酮治疗组(A组)、阿托伐他汀治疗组(B组)、吡格列酮治疗组(C组);另10只为正常对照组(对照组)。干预治疗6周后杀检,检测各组空腹血糖(FPG)、尿素氮(BUN)、血肌酐(SCr)、血脂及尿肌酐清除率(Ccr),用ELISA法检测肾组织超氧化物岐化酶(SOD)、丙二醛(MDA)、一氧化氮合酶(NOS)。结果与对照组比较,各组FPG、BUN、血脂、Ccr、SOD、MDA、NOS升高,SCr降低,均以T2DM组明显(P<0.05或<0.01);与B、C组比较,A组Ccr、LDL-C、NOS、MDA降低,SOD升高(P<0.05或<0.01)。结论皮甾酮能降低T2DM大鼠的Ccr,其可能通过减少肾组织MDA、NOS,升高SOD发挥抗氧化效应,因而具有防治DKD的作用。     

Pramlintide reduced markers of oxidative stress in the postprandial period in patients with type 2 diabetes

BACKGROUND: The production of oxidative stress as a result of postprandial hyperglycaemia is now recognized as an important contributing factor in the development of diabetes complications. The objective of this study was to examine the effects of pramlintide on plasma concentrations of glucose and several markers of oxidative stress in patients with type 2 diabetes following a standardized meal. METHODS: This was a randomized, single-blind, placebo-controlled, crossover study conducted at two clinical research centres in the United States. A total of 19 subjects (9 men and 10 women) with type 2 diabetes using mealtime insulin participated in the study. Pramlintide (120 microg), or placebo, and rapid-acting mealtime insulin were administered prior to a standardized meal on two separate study days. Plasma concentrations of glucose, nitrotyrosine (NT), oxidized-LDL cholesterol (OxLDL-C), and total radical trapping parameter (TRAP) were assessed during the 4-h postprandial period. RESULTS: Compared to placebo, pramlintide treatment reduced postprandial excursions of glucose, NT, and OxLDL-C and protected TRAP from consumption. Correlation analysis revealed positive associations between placebo-corrected glucose incremental AUC(0-4 h) and both NT and OxLDL-C and a negative association between placebo-corrected glucose incremental AUC(0-4h) and TRAP. CONCLUSIONS: The reduction in postprandial glucose excursions achieved with addition of pramlintide to rapid-acting insulin in type 2 diabetes was associated with a reduction in postprandial markers of oxidative stress.     

Formation and characterization of an all-ferrous Rieske cluster and stabilization of the [2Fe-2S]0 core by protonation

The all-ferrous Rieske cluster, [2Fe-2S]⁰, has been produced in solution and characterized by protein-film voltammetry and UV–visible, EPR, and Mössbauer spectroscopies. The [2Fe-2S]⁰ cluster, in the overexpressed soluble domain of the Rieske protein from the bovine cytochrome bc₁ complex, is formed at –0.73 V at pH 7. Therefore, at pH 7, the [2Fe-2S]^1+/0 couple is 1.0 V below the [2Fe-2S]^2+/1+ couple. The two cluster-bound ferrous irons are both high spin (S = 2), and they are coupled antiferromagnetically (–J ≥ 30 cm^–1, H =–2JS1·S2) to give a diamagnetic (S = 0) ground state. The ability of the Rieske cluster to exist in three oxidation states (2+, 1+, and 0) without an accompanying coupled reaction, such as a conformational change or protonation, is highly unusual. However, uncoupled reduction to the [2Fe-2S]⁰ state occurs at pH > 9.8 only, and at high pH the intact cluster persists in solution for <1 min. At pH < 9.8, the all-ferrous cluster is stabilized significantly by protonation. A combination of experimental data and calculations based on density functional theory suggests strongly that the proton binds to one of the cluster μ₂-sulfides, consistent with observations that reduced [3Fe-4S] clusters are protonated also. The implications for our understanding of coupled reactions at iron–sulfur clusters and of the factors that determine the relative stabilities of their different oxidation states are discussed.Iron–sulfur (FeS) clusters are essential to all forms of life. Most frequently, they are simple electron carriers, but they also constitute catalytic centers, structural scaffolds, and sensors, and they undergo oxidative degradation (1, 2). The rhombic [2Fe-2S], cuboidal [3Fe-4S], and cubane [4Fe-4S] clusters are the most common, but elaboration of these basic modules has produced clusters that contain heterometals and up to eight iron centers. For example, the catalytic clusters in acetyl-CoA synthase contain nickel, and the P-cluster and iron–molybdenum cofactor of nitrogenase can be considered to be two cuboidal subclusters joined by sulfurs. Assembly, disassembly, and interconversion of the simpler clusters are exploited in oxygen sensing and in the control of intracellular iron levels, and they also constitute enzyme active sites, such as in aconitase, chloroplast ferredoxin:thioredoxin reductase, and biotin synthase. In contrast, uncontrolled cluster disassembly (during oxidative stress) accelerates the production of reactive oxygen species and therefore is potentially very damaging.Formally, each iron center in a cluster can be ferric or ferrous, so [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters have three, four, and five possible oxidation states, respectively. In proteins, the oxidation states of [3Fe-4S] clusters cover the widest range because many have been observed in the 1+ (all-ferric), 0, and 2–(all-ferrous) states (3). Formation of the [3Fe-4S]⁰ state is associated with protonation (4), and reduction to the 2–state occurs only upon the uptake of a total of three protons (3). Therefore, the overall charge is conserved, and it is likely that [3Fe-4S] clusters exist in more than two oxidation states because they can be protonated. The protons probably bind on the three μ₂-sulfides, consistent with the ability of [3Fe-4S]⁰ clusters to coordinate a fourth metal ion (5). In contrast, most [4Fe-4S] clusters in proteins are confined to only two oxidation states, either the 2+ and 1+ states or the 3+ and 2+ states in high-potential iron proteins. The [4Fe-4S] cluster in the Fe-protein of nitrogenase (“the Fe-protein”) is unique because it can exist in the all-ferrous state and is stable in more than two oxidation states (2+,1+, and 0) (6). This versatility may be because of its unusually high solvent accessibility (7), although during turnover it undergoes significant conformational changes coupled to nucleotide binding (8), and protonation of the all-ferrous state has not been excluded. The nitrogenase [8Fe-7S] P-cluster (“the P cluster”), the only high-nuclearity cluster not involved directly in small molecule activation, also exists in three oxidation states, but interconversions between them are coupled to changes in cluster structure and ligation and to protonation; the most reduced state may be all-ferrous (9, 10). There is no confirmed example of an FeS cluster in a protein undergoing sequential uncoupled redox transformations.Under physiological conditions, [2Fe-2S] clusters have been observed in the 2+ and 1+ states only. The all-ferrous state may not have been observed because the potential for its formation is too negative, the all-ferrous cluster is unstable, or two-electron reduction is precluded in the absence of a charge-compensation mechanism. In contrast, in anaerobic nonaqueous solvents, synthetic [2Fe-2S] analogues can be reduced sequentially by two electrons (11); the reduction potentials are reported to be only ≈0.25 V apart, but the proposed all-ferrous nature of the fully reduced state has not been demonstrated. A [2Fe-2S]⁰ cluster was generated artificially in spinach ferredoxin by irreversible complexation of the protein to a chromium reductant, increasing the reduction potential significantly and adding extra positive charge (12). Voltammetric signals from the soluble Rieske domain from the bovine heart cytochrome bc₁ complex (–0.84 V at pH 7, potential reported to be pH independent) were attributed to formation of the all-ferrous cluster, but no characterization was attempted (13). Here, we describe the reversible formation of a stable, unmodified [2Fe-2S]⁰ cluster-containing protein and its extensive characterization. The [2Fe-2S]⁰ cluster is a Rieske cluster, so its formation is facilitated by the neutral, electronegative histidine ligands. Our results provide insight into the potentials, stabilities, and reactivities of the different oxidation states of the simplest FeS clusters and are also relevant to understanding the structures and functions of higher nuclearity clusters.     

Effects of hypoxia reoxygenation in brain slices from rats with type 1-like diabetes mellitus

BACKGROUND: The aim of this study was to determine whether the brain tissue of type 1 diabetic animals is more susceptible to damage by hypoxia reoxygenation than healthy animals. METHODS: This study used rats with diabetes of 1, 2 and 3 months (N = 15 rats/group). Brain slices were subjected to hypoxia and reoxygenation for 180 min in vitro. We measured oxidative stress (lipid peroxidation, glutathione concentration and enzyme activities related to glutathione), concentration of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) pathway (nitrite + nitrate, activities of constitutive (cNOS) and inducible (iNOS) nitric oxide synthase). As a parameter of cell death we measured the efflux of lactate dehydrogenase (LDH). RESULTS: After reoxygenation LDH activity increased in comparison to nondiabetic animals by 40, 40.6 and 68.9% in animals with diabetes of 1, 2 and 3 months duration, respectively. These changes were accompanied by greater increases in lipid peroxides (25.4, 93.7 and 92.8%). PGE(2) accumulated in significantly larger amounts in diabetic animals (62.5, 85.5 and 114%), and nitrite + nitrate accumulation was significantly greater in rats with diabetes of 2 (40.2%) and 3 months duration (24.0%). iNOS activity increased significantly in all the groups of diabetic animals, with the largest increases in rats with diabetes of 2 (18.6%) and 3 months duration (21.1%). CONCLUSIONS: The biochemical pathways involved in oxidative stress and neuronal death are more sensitive to hypoxia reoxygenation in type 1-like diabetic, as compared to normal, rats.     

Impact of diabetes mellitus on the relationships between iron-, inflammatory- and oxidative stress status

BACKGROUND: Diabetes is an inflammatory condition associated with iron abnormalities and increased oxidative damage. We aimed to investigate how diabetes affects the interrelationships between these pathogenic mechanisms. METHODS: Glycaemic control, serum iron, proteins involved in iron homeostasis, global antioxidant capacity and levels of antioxidants and peroxidation products were measured in 39 type 1 and 67 type 2 diabetic patients and 100 control subjects. RESULTS: Although serum iron was lower in diabetes, serum ferritin was elevated in type 2 diabetes (p = 0.02). This increase was not related to inflammation (C-reactive protein) but inversely correlated with soluble transferrin receptors (r = - 0.38, p = 0.002). Haptoglobin was higher in both type 1 and type 2 diabetes (p < 0.001) and haemopexin was higher in type 2 diabetes (p < 0.001). The relation between C-reactive protein and haemopexin was lost in type 2 diabetes (r = 0.15, p = 0.27 vs r = 0.63, p < 0.001 in type 1 diabetes and r = 0.36, p = 0.001 in controls). Haemopexin levels were independently determined by triacylglycerol (R(2) = 0.43) and the diabetic state (R(2) = 0.13). Regarding oxidative stress status, lower antioxidant concentrations were found for retinol and uric acid in type 1 diabetes, alpha-tocopherol and ascorbate in type 2 diabetes and protein thiols in both types. These decreases were partially explained by metabolic-, inflammatory- and iron alterations. An additional independent effect of the diabetic state on the oxidative stress status could be identified (R(2) = 0.5-0.14). CONCLUSIONS: Circulating proteins, body iron stores, inflammation, oxidative stress and their interrelationships are abnormal in patients with diabetes and differ between type 1 and type 2 diabetes.     

Differential gene expression of NADPH oxidase (p22phox) and hemoxygenase-1 in patients with Type 2 diabetes and microangiopathy.

AIMS: While the downstream effects of increased reactive oxygen species (ROS) in the pathogenesis of diabetes were well studied, only a few studies have explored the cellular sources of ROS. We examined whether protection against oxidative stress is altered in patients with diabetes and microangiopathy by examining changes in NADPH oxidase (p22(phox)) and hemoxygenase-1 (HO-1) levels. METHODS: NADPH oxidase (p22(phox)) and HO-1 gene expression were probed by RT-PCR using leucocytes from patients with Type 2 diabetes without (n = 19) and with microangiopathy (n = 20) and non-diabetic subjects (n = 17). Levels of lipid peroxidation as measured by thiobarbituric reactive substances (TBARS) and protein carbonyl content (PCO) were determined by fluorimetric and spectrophotometric methods, respectively. RESULTS: p22(phox) gene expression (mean +/- SE) was significantly (P < 0.05) higher in diabetic patients with (0.99 +/- 0.04) and without microangiopathy (0.86 +/- 0.05) compared with control subjects (0.66 +/- 0.05). Consistent with the mRNA data, the p22(phox) protein expression and NADPH oxidase activity was also increased in cells from diabetic patients compared with control subjects. However, HO-1 gene expression was significantly (P < 0.05) lower in patients with (0.73 +/- 0.03) and without microangiopathy (0.85 +/- 0.02) compared with control subjects (1.06 +/- 0.03). The mean (+/- SE) levels of TBARS were significantly (P < 0.05) higher in diabetic patients with (14.36 +/- 1.3 nM/ml) and without microangiopathy (12.20 +/- 1.3 nM/ml) compared with control subjects (8.58 +/- 0.7 nM/ml). The protein carbonyl content was also significantly (P < 0.05) higher in diabetic patients with (1.02 +/- 0.04 nmol/mg protein) and without microangiopathy (0.84 +/- 0.06 nmol/mg protein) compared with control subjects (0.48 +/- 0.02 nmol/mg protein). In diabetic subjects, increased p22(phox) gene expression was negatively correlated with HO-1 and positively correlated with TBARS, PCO, HbA(1c) and diabetes duration. In contrast, HO-1 gene expression was correlated negatively with p22phox, TBARS, PCO, HbA(1c) and diabetes duration. CONCLUSION: Our results indicate that increased oxidative damage is seen in Asian Indians with Type 2 diabetes and microangiopathy and is associated with increased NADPH oxidase (p22(phox)) and decreased HO-1 gene expression.     

Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli.

A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Th?gersen [Nagai, K. & Th?gersen, M. C. (1984) Nature (London) 309, 810-812]. Expression was under control of the lambda PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage lambda cII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO4/PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S] center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450scc and P-450(11) beta and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function.