Maturation of embryonic chick liver delta-aminolevulinate synthase: precursor pools and regulation by intra-cellularly produced heme

1. Immunoblot analyses were carried out to determine the relative distributions of delta-aminolevulinate synthase (ALA synthase) in mitochondrial and cytosol fractions prepared from embryos at different times after injections with allylisopropylacetamide (AIA). 2. The results indicated that the molecular mass of mature ALA synthase (Mr 65,000) increased with time in mitochondria. 3. At no time was the precursor form (Mr 75,000) of the enzyme detected either in mitochondria or in the cytosol. 4. In primary cultures of hepatocytes, where the increased production of ALA synthase had been induced with AIA, addition of delta-aminolevulinic acid (ALA) and Fe2(SO4)3 into the culture medium completely blocked the processing of the precursor form of the enzyme. 5. On the other hand, the addition of ALA together with deferoxamine mesylate into the medium had no detectable effect on the maturation of ALA synthase in the hepatocytes. 6. The results indicated: first, that upon induction of porphyria the pools of pre-ALA synthase in liver are relatively low in chick embryos when compared with those in other organisms; and second, that increased heme production by the hepatocytes caused the inhibition of processing of the precursor form of ALA synthase.     

delta-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals.

delta-Aminolaevulinate (ALA) synthase, the rate-limiting enzyme in haem biosynthesis in the normal liver, was examined in human HepG2 hepatoma cells. Haemin, up to 100 microM, had no effect on ALA synthase activity in vitro; it did, however, exhibit a dose-dependent inhibitory action when added to cells growing in culture (half-maximal inhibition at 1 microM). The half-life of ALA synthase activity after haemin treatment was 2 h, which was similar to that found after treatment with cycloheximide. Cells treated with actinomycin D showed a longer half-life of the enzyme activity, i.e. 4 h, compared with haemin or cycloheximide treatment. Treatment of cells with succinylacetone markedly inhibited the activity of ALA dehydratase and 59Fe incorporation into haem, but in increased ALA synthase activity. Both the haemin-induced repression and the succinylacetone-mediated de-repression of ALA synthase activity were reversible within 4 h after replacing the medium with fresh medium without the chemical. In addition to succinylacetone, dimethyl sulphoxide and 3-methylcholanthrene induced the enzyme. Induction of ALA synthase by these chemicals was also suppressed by treatment of cells with haemin. These findings indicate that the level of ALA synthase in HepG2 cells is maintained by both synthesis and degradation of the enzyme, and that the synthesis of the enzyme is regulated by the concentration of regulatory free haem in the cell.     

Biogenesis of embryonic chick liver delta-aminolevulinate synthase: regulation of the level of mRNA by hemin

The effects of hemin on the concentration of the mRNA for delta-aminolevulinate synthase (ALA synthase) and on the association of the messenger with polysomes were investigated in primary cultures of embryonic chick hepatocytes incubated with allylisopropylacetamide (AIA). A synthetic 24-mer DNA complementary to ALA synthase mRNA was used to determine by solution hybridization the effects of AIA and of AIA plus hemin on the ALA synthase-specific RNA sequences in the cells. The results indicated that ALA synthase mRNA concentrations increased significantly in hepatocytes incubated for 5 h with AIA (0.075 mg/ml), and that hemin in the medium (2 or 10 microM) blocked the increase in the messenger. When delta-aminolevulinic acid (ALA) and FeCl3 were added into the culture medium (1 mM and 5 microM, respectively), the increase in ALA synthase mRNA brought on by AIA was also inhibited. Neither ALA nor FeCl3, when individually added to the cultures, was as effective as the combination of the two. The results with ALA + FeCl3 suggested that stimulation of intracellular production of heme was also effective in blocking the increase in ALA synthase mRNA caused by AIA. Finally, the distributions of ALA synthase mRNA were compared in polysomes isolated from hepatocytes which had been incubated with AIA for 5 h in the presence and absence of 10 microM hemin in the medium. Although a drop was detected in the concentration of ALA synthase mRNA in polysomes from hepatocytes incubated with hemin for 30 min, the decrease was explained by the effect of hemin on the mRNA concentration in the cells.     

Regulation of biogenesis of liver delta-aminolevulinate synthase: effects of structural modifications of heme on the enzyme's RNA

1. The aim of this study was to determine the effects of several metallo-porphyrins, derived by modifications of heme, on the concentration delta-aminolevulinate (ALA) synthase RNA in hepatocytes. 2. Primary cultures of chick embryo hepatocytes were incubated with allylisopropylacetamide (AIA) for 5 hr in the presence and absence of each metallo-porphyrin (10 microM). At the end of each incubation, total RNA was isolated from the cells and analyzed for ALA synthase-specific RNA by solution hybridization. 3. The concentration of ALA synthase RNA increased 7.3 fold in hepatocytes incubated with AIA alone. The AIA-induced elevations in the enzyme's RNA were blocked partially and equally in cells. incubated with zinc- or with iron-protoporphyrin IX. The block was greater in cells incubated with cobalt-protoporphyrin IX. 4. Modifications of the side chains of the porphyrin ring at positions 2 and 4, giving mesoporphyrin IX and deuteroporphyrin IX, changed the effectiveness of the iron- and the cobalt-porphyrins to limit the AIA-induced increase in ALA synthase RNA. The modifications did not affect the capacities of the zinc-porphyrins to inhibit the rise in RNA. 5. In conclusion, the effect of a given metallo-porphyrin on liver ALA synthase RNA following side chain modification depended on the coordinated metal.     

Regulation of production of delta-aminolaevulinate synthase in tissues of chick embryos. Effects of porphyrogenic agents and of haem precursors.

Studies conducted by several groups have established that porphyrogenic agents which caused elevations in chick-embryo liver delta-aminolaevulinate (ALA) synthase activity also increased the concentrations of the enzyme's RNA, and that haemin inhibited these elevations. We have determined in this study, using immune-blot analyses, that administration in ovo of allylisopropylacetamide (AIA) in combination with diethyl 1,4-dihydro-2,4,6-trimethyl,3,5-pyridinedicarboxylate (DDC) increased the mass of ALA synthase in intestine and kidney of chick embryos. Furthermore, the molecular mass of the subunit of the enzyme in those tissues appeared identical with that of liver ALA synthase. Using a synthetic oligonucleotide complementary to ALA synthase mRNA, we determined by solution hybridization and Northern-blot analyses that AIA and DDC also increased the concentrations of ALA synthase mRNA in intestine and kidney and that testosterone elevated the concentration of the RNA in kidney. In analyses of RNA obtained from chick-embryo liver, intestine, kidney, heart, brain and lung, the probe bound primarily in each case to a single 2.3 kb RNA. Finally, the haem precursors ALA and FeCl3, when injected together into the fluid surrounding embryos, inhibited both the elevations in ALA synthase mass and RNA concentration brought about by porphyrogenic agents in liver, kidney and intestine. Thus the results indicated that: (1) certain porphyrogenic agents increased ALA synthase mass and RNA in chick-embryo intestine and kidney, in addition to liver; (2) ALA and FeCl3 inhibited the elevations; and (3) the sizes of ALA synthase's subunit as well as the enzyme's mRNA appeared identical, in each case, in all tissues examined.     

Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms

Regulation of delta-aminolevulinic acid (ALA) synthase and heme oxygenase was analyzed in primary rat hepatocytes and in two immortalized cell lines, CWSV16 and CWSV17 cells. ALA synthase was induced by 4,6-dioxohepatnoic acid (4,6-DHA), a specific inhibitor of ALA dehydratase, in all three systems; however, the induction in CWSV17 cells was greater than in either of the other two systems. Therefore, CWSV17 cells were used to explore the regulation of both enzymes by heme and 4,6-DHA. Data obtained from detailed concentration curves demonstrated that 4,6-DHA induced the activity of ALA synthase once ALA dehydratase activity became rate-limiting for heme biosynthesis. Heme induced heme oxygenase activity with increases occurring at concentrations of 10 microM or greater. Heme blocked the 4,6-DHA-dependent induction of ALA synthase with an EC50 of 1.25 microM. Heme-dependent decreases of ALA synthase mRNA levels occurred more quickly and at lower concentrations than heme-dependent increases of heme oxygenase mRNA levels. ALA synthase mRNA remained at reduced levels for extended periods of time, while the increases in heme oxygenase mRNA were much more transient. The drastic differences in concentrations and times at which heme-dependent effects were observed strongly suggest that two-different heme-dependent mechanisms control the ALA synthase and heme oxygenase mRNAs. In CWSV17 cells, heme decreased the stability of ALA synthase mRNA from 2.5 to 1.3 h, while 4,6-DHA increased the stability of the mRNA to 5.2 h. These studies demonstrate that regulation of ALA synthase mRNA levels by heme in a mammalian system is mediated by a change in ALA synthase mRNA stability. The results reported here demonstrate the function of the regulatory heme pool on both ALA synthase and heme oxygenase in a mammalian hepatocyte system.     

Regulation of production of embryonic chick liver delta-aminolevulinate synthase: effects of testosterone and of hemin on the mRNA of the enzyme

The effects of testosterone and of hemin on the concentration of the mRNA of embryonic chick liver ALA synthase were investigated. Using cDNA-RNA liquid hybridization analyses, we determined that testosterone, when injected into the fluid surrounding chick embryos, caused a dose-dependent increase in the concentration of ALA synthase mRNA in liver. Similarly, addition of testosterone (5 micrograms/ml) or of 75 micrograms/ml of allylisopropylacetamide (AIA) into the medium of chick embryo hepatocytes maintained in culture caused an increase in the concentration of ALA synthase mRNA. Hemin (2 or 5 microM), when added to the culture medium, inhibited the elevations of ALA synthase mRNA concentration brought on by testosterone and by AIA.     

On the mechanism of the okadaic acid-induced inhibition of phosphatidylcholine biosynthesis in isolated rat hepatocytes.

The mechanism of inhibition of phosphatidylcholine biosynthesis by okadaic acid was investigated in suspension cultures of isolated rat hepatocytes. Cells were pulsed with [methyl-3H]choline and chased in the absence or presence of 1 microM okadaic acid for up to 120 min. Phosphatidylcholine biosynthesis was inhibited after 15 min of chase. To see if okadaic acid altered the degree of phosphorylation of cytidylyltransferase (CT), hepatocytes were incubated with 32P(i) and chased in the absence or presence of okadaic acid. Okadaic acid caused a rapid (within 15 min) increase in the phosphorylation state of the cytosolic enzyme. Two-dimensional peptide map analysis revealed an increase in the phosphorylation of several peptides in okadaic acid-treated hepatocytes compared with controls. After 15 min of incubation of hepatocytes with okadaic acid, membrane CT activity was decreased and a corresponding increase in cytosolic CT activity was observed. In hepatocytes incubated with okadaic acid and oleate a correlation between membrane CT activity, diacylglycerol level, and phosphatidylcholine biosynthesis was observed. These data suggest that the concentration of diacylglycerol is responsible for the increase in membrane CT activity and subsequently phosphatidylcholine biosynthesis in oleate-treated cells. We postulate that the okadaic acid-induced decrease in phosphatidylcholine biosynthesis is due to an increase in the phosphorylation state of CT which promotes a translocation of CT activity from the membranes to the cytosol.     

Biogenesis of liver delta-aminolevulinate synthase. The role of cAMP in the induction

In primary cultures of chick embryo hepatocytes pulse labeled with [35S]methionine, immunochemical analyses indicated that adenosine 3':5'-cyclic monophosphate (cAMP) did not affect either the rate of production or the maturation of delta-aminolevulinate synthase (ALA synthase). In addition, allylisopropylacetamide caused a slight drop in intracellular cAMP while testosterone caused the levels of cAMP to rise to 260% of the basal levels measured in hepatocytes in culture. Thus the results of this study did not indicate a direct short-term role for cAMP in the regulation of production of ALA synthase.     

Separation and partial characterization of enzymes catalyzing delta-aminolevulinic acid formation in Synechocystis sp. PCC 6803

Formation of the universal tetrapyrrole precursor, delta-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differentiatial ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2',5'-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 microM, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 microM. GSA aminotransferase was inhibited 50% by 0.5 microM gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 degrees C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.     

Effect of endogenous heme generation on delta-aminolevulinic acid synthase activity in rat liver mitochondria.

This study examined the possibility that generation of heme within mitochondria may provide a local concentration sufficient to inhibit the activity of delta-aminolevulinic acid (ALA) synthase, the enzyme that catalyzes the rate-limiting step in hepatic heme biosynthesis. This was accomplished by simultaneously running ALA synthase and heme synthase activities in intact mitochondria isolated from rat liver. Radiochemical assays were used to measure the enzyme activities. ALA synthase activity did not decrease as the rate of heme formation was increased by varying the concentration of substrates for heme synthase. Even at a rate of heme generation estimated to be at least 75 times the rate occuring in vivo, ALA synthase activity was unchanged. We conclude that end product inhibition of ALA synthase activity by heme is not an important physiological mechanism for regulation of hepatic heme biosynthesis.     

Homocitrate synthase from Penicillium chrysogenum. Localization, purification of the cytosolic isoenzyme, and sensitivity to lysine.

Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 +/- 10 kDa as determined by gel filtration and a pH of 4.9 +/- 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176.     

Coordinate elevations of liver delta-aminolevulinate synthase and cytochrome P-450 RNA by phenobarbital in chicken embryos: the effects of heme

1. The role of heme in the coordinate elevations of liver delta-aminolevulinate (ALA) synthase activity and microsomal cytochrome P-450 concentration induced by phenobarbital (PB) was investigated in the chicken embryo. 2. Eighteen day old chicken embryos were given PB, and the changes in liver content of PB-inducible cytochrome P-450 RNA and of ALA synthase RNA were determined at different times after exposure to the drug. 3. The concentrations of both types of RNA increased rapidly after PB administration, and by 9 hr the level of ALA synthase RNA was 55-fold higher than control and that of cytochrome P-450 RNA was 7-fold higher than normal. 4. While the rate of increase in ALA synthase activity paralleled closely that of the enzyme's RNA concentration, the rate of increase of spectrally active cytochrome P-450 concentration in microsomes lagged behind that of the apoprotein's RNA by several hours. 5. To test whether heme depletion was responsible for the coordinate inductions of the two enzymes, embryos were loaded with ALA 2 hr before exposure to PB. 6. The protocol led to a drop in the PB-inducible ALA synthase RNA concentration and to an increase in that of cytochrome P-450 RNA, measured 6 hr after drug administration. 7. In primary cultures of hepatocytes, hemin in the culture medium caused a modest drop in ALA synthase RNA concentration but had a variable effect on that of cytochrome P-450 RNA in cells incubated with PB for 9 hr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of porphyrin biosynthesis by exogenous 5-aminolevulinic acid in an aerobic photosynthetic bacterium, Erythrobacter sp. OCh 114

Exogenously administered 5-aminolevulinic acid (ALA) inhibited the formation of bacteriochlorophyll a (Bchl a) in a dose-dependent manner in the aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114, under dark growth conditions. The ALA concentration required for half-inhibition after 24-h growth was estimated to be about 3.0 mM. Porphyrin and Bchl precursors were not found in either the cells or the growth medium. The same inhibition was also observed with cytochrome c formation. When ALA was incubated with intact cells, a large amount of ALA was converted to an unknown metabolite. The pH optimum of the conversion was 7.8. The metabolite did not react with Ehrlich's reagent, but did so with ninhydrin, giving a yellow color. Based on analyses by several techniques including mass spectrometry, ir spectrometry, and paper electrophoresis, it was identified as 4-hydroxy-5-aminovaleric acid (HAVA). Authentic HAVA prepared from ALA was a competitive inhibitor of the enzyme, porphobilinogen synthase of Erythrobacter. The Ki value for authentic HAVA was calculated to be 2.4 mM from a Dixon plot and the HAVA concentration required for half-inhibition was 17 mM. It is concluded that in Erythrobacter cells, exogenous ALA is converted to the metabolite, HAVA, which is responsible for the inhibition of porphobilinogen synthase as well as that of Bchl a and cytochrome formation.     

Characterization of chicken liver L-alanine:4,5-dioxovaleric acid aminotransferase

1. A procedure is described for purifying the enzyme L-alanine:4,5-dioxovaleric acid aminotransferase (DOVA transaminase) from chicken liver. The enzyme catalyzes a transamination reaction between L-alanine and 4,5-dioxovaleric acid (DOVA), yielding delta-aminolevulinic acid (ALA). 2. In cell fractionation studies, DOVA transaminase activities were detected in mitochondria and in the post-mitochondrial supernatant fraction from liver homogenates. 3. For the mitochondrial enzyme, any of most L-amino acids could serve as a source for the amino group transferred to DOVA, but L-alanine appeared the preferred substrate. At pH 7.0, the enzyme had an apparent Km of 60 microM for DOVA and of 400 microM for L-alanine. 4. The enzyme was purified from disrupted mitoplasts in three steps: chromatography on DEAE-Sephacel, gel filtration through Sephadex G-150, and chromatography on hydroxyapatite. The yield was approx. 100 micrograms of enzyme protein per 10 g wet wt of liver. 5. The purified enzyme had a subunit mol. wt of 63,000 as determined by gel electrophoresis under denaturing conditions. 6. The activity of DOVA transaminase was also measured in embryonic chicken liver, and based on activity, the enzyme's capacity to produce ALA was significantly greater than that of ALA synthase. Unlike ALA synthase, however, DOVA transaminase activity did not increase in liver mitochondria of chicken embryos exposed for 18 hr to two potent porphyrogenic agents.     

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Induction of aminolevulinate synthase and porphyrins in cultured liver cells maintained in chemically defined medium. Permissive effects of hormones on induction process.

Primary liver cells, isolated from 16- 17-day-old chick embryos, were incubated in a serum-free chemically defined medium (Ham's F12) supplemented with hormones for up to 6 days. The culture method also includes the complete removal of contaminating red cells before the initiation of culture. On the 2nd day in cluture, the level of amino-levulinate (ALA) synthase activity in response to allylisopropylacetamide (AIA) was increased 6-fold in cells grown in F12. Insulin, hydrocortisone, and triiodothyronine alone had no appreciable effects on ALA synthase levels. On the other hand, when added with AIA, insulin, insulin plus hydrocortisone, insulin plus hydrocortisone triiodothyronine increased ALA synthase levels 17-, 50-, 110-fold, respectively. The maximally induced levels of ALA synthase activity by AIA in the presence of insulin, hydrocortisone, and triiodothyronine were approximately 15 nmol of ALA/mg of protein/h, 37 degrees or 3 micronmol of ALA/g of tissue/h, 37 degrees, a value similar to that found in ovo or at least 5 times greater than that found in rat liver. The morphology of hepatocytes was maintained for at least 6 days in culture, although the induction of ALA synthase was reduced after the 4th day unless triiodothyronine was present. Dibutyryl adenosine 3':5'-monophosphate (10(8) M) or glucagon (5x10(8) M) had little effect on the induced as well as noninduced levels of ALA synthase or porphyrins. These data demonstrate a "permissive" effect of insulin, hydrocortisone, and triiodothyronine on the induction of ALA synthase and porphyrins by AIA in cultured chick embryo liver cells. In the absence of insulin hydrocortisone, or triiodothyronine, AIA produces only a slight increase in ALA synthase activity or porphyrins (or both); on the other hand, it produces a marked increase in the enzyme activity and porphyrins when these hormones are added to the culture medium. The term "permissive" is applied to these hormone-dependent effects. A sensitive spectrofluorometric method for heme quantitation allowed us to follow changes in the cellular heme content in hemoglobin-free cultured liver cells. Heme content in the cultured liver cells was approximately 250 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein at the initiation of culture but gradually declined to 175 pmol/mg of protein during 48 h of incubation. The apparent decrease in heme content may be accounted for by the concomitant increase in protein content in these cells.     

Fidelity of metal insertion into hydrogenases.

The fidelity of metal incorporation into the active center of hydrogenase 3 from Escherichia coli was studied by analyzing the inhibition of the maturation pathway by zinc and other transition metals. Hydrogenase maturation of wild-type cells was significantly affected only by concentrations of zinc or cadmium higher than 200 microM, whereas a mutant with a lesion in the nickel uptake system displayed a total blockade of the proteolytic processing of the precursor form into the mature form of the large subunit after growth in the presence of 10 microM Zn(2+). The precursor could not be processed in vitro by the maturation endopeptidase even in the presence of an excess of nickel ions. Evidence is presented that zinc does not interfere with the incorporation of iron into the metal center. Precursor of the large subunit accumulated in nickel proficient cells formed a transient substrate complex with the cognate endoprotease HycI whereas that of zinc-supplemented cells did not. The results show that zinc can intrude the nickel-dependent maturation pathway only when nickel uptake is blocked. Under this condition zinc appears to be incorporated at the nickel site of the large subunit and delivers a precursor not amenable to proteolytic processing since the interaction with the endoprotease is blocked.