Feedback Inhibition of Chlorophyll Synthesis in the Phytochrome Chromophore-Deficient aurea and yellow-green-2 Mutants of Tomato

The aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) are unable to synthesize the linear tetrapyrrole chromophore of phytochrome, resulting in plants with a yellow-green phenotype. To understand the basis of this phenotype, we investigated the consequences of the au and yg-2 mutations on tetrapyrrole metabolism. Dark-grown seedlings of both mutants have reduced levels of protochlorophyllide (Pchlide) due to an inhibition of Pchlide synthesis. Feeding experiments with the tetrapyrrole precursor 5-aminolevulinic acid (ALA) demonstrate that the pathway between ALA and Pchlide is intact in au and yg-2 and suggest that the reduction in Pchlide is a result of the inhibition of ALA synthesis. This inhibition was independent of any deficiency in seed phytochrome, and experiments using an iron chelator to block heme synthesis demonstrated that both mutations inhibited the degradation of the physiologically active heme pool, suggesting that the reduction in Pchlide synthesis is a consequence of feedback inhibition by heme. We discuss the significance of these results in understanding the chlorophyll-deficient phenotype of the au and yg-2 mutants.

Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary

We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet7]cyt b562, by thioester-mediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the α-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a full-length unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet7]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by ≈45 mV. The use of the 1-phenyl-2-mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Molecular Characterization of Photomixotrophic Tobacco Cells Resistant to Protoporphyrinogen Oxidase-Inhibiting Herbicides1

Peroxidizing herbicides inhibit protoporphyrinogen oxidase (Protox), the last enzyme of the common branch of the chlorophyll- and heme-synthesis pathways. There are two isoenzymes of Protox, one of which is located in the plastid and the other in the mitochondria. Sequence analysis of the cloned Protox cDNAs showed that the deduced amino acid sequences of plastidial and mitochondrial Protox in wild-type cells and in herbicide-resistant YZI-1S cells are the same. The level of plastidial Protox mRNA was the same in both wild-type and YZI-1S cells, whereas the level of mitochondrial Protox mRNA YZI-1S cells was up to 10 times the level of wild-type cells. Wild-type cells were observed by fluorescence microscopy to emit strong autofluorescence from chlorophyll. Only a weak fluorescence signal was observed from chlorophyll in YZI-1S cells grown in the Protox inhibitor N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide. Staining with DiOC6 showed no visible difference in the number or strength of fluorescence between wild-type and YZI-1S mitochondria. Electron micrography of YZI-1S cells showed that, in contrast to wild-type cells, the chloroplasts of YZI-1S cells grown in the presence of N-(4-chloro-2-fluoro-5-propagyloxy)-phenyl-3,4,5,6-tetrahydrophthalimide exhibited no grana stacking. These results suggest that the herbicide resistance of YZI-1S cells is due to the overproduction of mitochondrial Protox.

Magnesium-Chelatase from Developing Pea Leaves1 : Characterization of a Soluble Extract from Chloroplasts and Resolution into Three Required Protein Fractions

Mg-chelatase catalyzes the ATP-dependent insertion of Mg2+ into protoporphyrin-IX to form Mg-protoporphyrin-IX. This is the first step unique to chlorophyll synthesis, and it lies at the branch point for porphyrin utilization; the other branch leads to heme. Using the stromal fraction of pea (Pisum sativum L. cv Spring) chloroplasts, we have prepared Mg-chelatase in a highly active (1000 pmol 30 min−1 mg−1) and stable form. The reaction had a lag in the time course, which was overcome by preincubation with ATP. The concentration curves for ATP and Mg2+ were sigmoidal, with apparent Km values for Mg2+ and ATP of 14.3 and 0.35 mm, respectively. The Km for deuteroporphyrin was 8 nm. This Km is 300 times lower than the published porphyrin Km for ferrochelatase. The soluble extract was separated into three fractions by chromatography on blue agarose, followed by size-selective centrifugal ultrafiltration of the column flow-through. All three fractions were required for activity, clearly demonstrating that the plant Mg-chelatase requires at least three protein components. Additionally, only two of the components were required for activation; both were contained in the flow-through from the blue-agarose column.

Cytochrome c′ folding triggered by electron transfer: Fast and slow formation of four-helix bundles

Reduced (FeII) Rhodopseudomonas palustris cytochrome c′ (Cyt c′) is more stable toward unfolding ([GuHCl]1/2 = 2.9(1) M) than the oxidized (FeIII) protein ([GuHCl]1/2 = 1.9(1) M). The difference in folding free energies (ΔΔGf° = 70 meV) is less than half of the difference in reduction potentials of the folded protein (100 mV vs. NHE) and a free heme in aqueous solution (≈−150 mV). The spectroscopic features of unfolded FeII–Cyt c′ indicate a low-spin heme that is axially coordinated to methionine sulfur (Met-15 or Met-25). Time-resolved absorption measurements after CO photodissociation from unfolded FeII(CO)–Cyt c′ confirm that methionine can bind to the ferroheme on the microsecond time scale [kobs = 5(2) × 104 s−1]. Protein folding was initiated by photoreduction (two-photon laser excitation of NADH) of unfolded FeIII–Cyt c′ ([GuHCl] = 2.02–2.54 M). Folding kinetics monitored by heme absorption span a wide time range and are highly heterogeneous; there are fast-folding (≈103 s−1), intermediate-folding (102–101 s−1), and slow-folding (10−1 s−1) populations, with the last two likely containing methionine-ligated (Met-15 or Met-25) ferrohemes. Kinetics after photoreduction of unfolded FeIII–Cyt c′ in the presence of CO are attributable to CO binding [1.4(6) × 103 s−1] and FeII(CO)–Cyt c′ folding [2.8(9) s−1] processes; stopped-flow triggered folding of FeIII–Cyt c′ (which does not contain a protein-derived sixth ligand) is adequately described by a single kinetics phase with an estimated folding time constant of ≈4 ms [ΔGf° = −33(3) kJ mol−1] at zero denaturant.

On the molecular mechanism of chloroquine's antimalarial action.

Chloroquine is thought to exert its antimalarial effect by preventing the polymerization of toxic heme released during proteolysis of hemoglobin in the Plasmodium digestive vacuole. The mechanism of this blockade has not been established. We incubated cultured parasites with subinhibitory doses of [3H]chloroquine and [3H] quinidine. These [3H]quinoline compounds became associated with hemozoin as assessed by electron microscope autoradiography and subcellular fractionation. In vitro, binding of [3H]quinoline inhibitors to the hemozoin chain depended on the addition of heme substrate. These data counter previous conclusions regarding the lack of quinoline association with hemozoin, explain the exaggerated accumulation of quinolines in the plasmodium digestive vacuole, and suggest that a quinoline heme complex incorporates into the growing polymer to terminate chain extension, blocking further sequestration of toxic heme.

Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.

We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal-transduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine.

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

The kinetics of photo-induced electrontransfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferarfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of 4.8 x 10(7) M(-1) x s(-1). The limiting value of kobs at high HiPIP concentration is 95 s(-1). The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is approximately 3 x 10(5) s(-1). This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 microM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 10(6) M(-1) x s(-1) and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferar fermentans HiPIP is a very effective electron donor to the photosynthetic RC.

X-ray structure determination of a metastable state of carbonmonoxy myoglobin after photodissociation.

The x-ray structure of carbon monoxide (CO)-ligated myoglobin illuminated during data collection by a laser diode at the wavelength lambda = 690 nm has been determined to a resolution of 1.7 A at T = 36 K. For comparison, we also measured data sets of deoxymyoglobin and CO-ligated myoglobin. In the photon-induced structure the electron density associated with the CO ligand can be described by a tube extending from the iron into the heme pocket over more than 4 A. This density can be interpreted by two discrete positions of the CO molecule. One is close to the heme iron and can be identified to be bound CO. In the second, the CO is dissociated from the heme iron and lies on top of pyrrole ring C. At our experimental conditions the overall structure of myoglobin in the metastable state is close to the structure of a CO-ligated molecule. However, the iron has essentially relaxed into the position of deoxymyoglobin. We compare our results with those of Schlichting el al. [Schlichting, I., Berendzen, J., Phillips, G. N., Jr., & Sweet, R. M. (1994) Nature 317, 808-812], who worked with the myoglobin mutant (D122N) that crystallizes in the space group P6 and Teng et al. [Teng, T. Y., Srajer, V. & Moffat, K. (1994) Nat. Struct. Biol. 1, 701-705], who used native myoglobin crystals of the space group P2(1). Possible reasons for the structural differences are discussed.

A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Heterometallic hybrids of homometallic human hemoglobins.

Hybridization experiments between normal Hb tetramers (Fe2+ Hb) and those with four metal-substituted hemes (i.e., replacement of Fe2+ by Co2+, Mg2+, Mn2+, Mn3+, Ni2+, or Zn2+) have revealed unexpected behavior. These homometallic Hbs have previously served as models that mimic the deoxy or oxy properties of normal Fe2+ Hb. In this study, hybrids were composed of one alpha 1 beta 1 dimer that is metal-substituted at both hemes, in association with a second dimer alpha 2 beta 2 that has normal Fe2+ hemes. Both metal-substituted subunits are unligated, whereas the two Fe2+ subunits either are both unligated or both ligated with O2, CO, or CN. It was found that four of the metal-substituted Hbs (Mg2+ Hb, Mn2+ Hb, Ni2+ Hb, and Zn2+ Hb) did not form detectable amounts of heterometallic hybrids with normal Fe2+ Hb even though (i) their homometallic parents formed tight tetrameric complexes with stabilities similar to that of Fe2+ Hb and (ii) hybrids with metal substitution at both alpha sites or both beta sites are known to form readily. This striking positional effect was independent of whether the normal Fe2+ hemes were ligated and of which ligand was used. These findings indicate that surprisingly large changes in tetramer behavior can arise from small and subtle perturbations at the heme sites. Possible origins of these effects are considered.