Novel Isonitrile Hydratase Involved in Isonitrile Metabolism

We previously discovered N-substituted formamide deformylase (NfdA) in Arthrobacter pascens F164, which degrades N-substituted formamide (Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., and Kobayashi, M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 13726–13731). In this study, we found an enzyme involved in the first step of isonitrile metabolism, isonitrile hydratase, that hydrates isonitrile to the corresponding N-substituted formamide. First, we investigated the optimum culture conditions for the production of isonitrile hydratase. The highest enzyme activity was obtained when A. pascens F164 was cultured in a nutrient medium containing N-benzylformamide. This Arthrobacter isonitrile hydratase was purified, characterized, and compared with Pseudomonas putida N19-2 isonitrile hydratase (InhA), which is the sole one reported at present. Arthrobacter isonitrile hydratase was found to have a molecular mass of about 530 kDa and to consist of 12 identical subunits. The apparent K_m value for cyclohexyl isocyanide was 0.95 ± 0.05 mm. A. pascens F164 grew and exhibited the isonitrile hydratase and N-substituted formamide deformylase activities when cultured in a medium containing an isonitrile as the sole carbon and nitrogen sources. However, both enzyme activities were not observed on culture in a medium containing glycerol and (NH₄)₂SO₄ as the sole carbon and nitrogen sources, respectively. These findings suggested that the Arthrobacter enzyme is an inducible enzyme, possibly involved in assimilation and/or detoxification of isonitrile. Moreover, gene cloning of the Arthrobacter enzyme revealed no sequence similarity between this enzyme and InhA. Comparison of their properties and features demonstrated that the two enzymes are biochemically, immunologically, and structurally different from each other. Thus, we discovered a new isonitrile hydratase named InhB.     

Isonitrile hydratase from Pseudomonas putida N19-2. Cloning,sequencing, gene expression,and identification of its active acid residue

Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19-2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.     

Clean oxidation of thiolates to sulfinates in a four-coordinate CoIII complex with a mixed carboxamido N-thiolato S donor set: relevance to nitrile hydratase

A new [Co(N2(SO2)2)(CNtBu)2](Et4N) complex 6 was prepared from N,N'-(3-mercapto-3-methyl-butyryl)-o-phenylenediamine and completely characterized. While the starting square planar complex [Co(N2S2)](Et4N) 4 was destroyed by dioxirane, the Co ligated thiolates of the six-coordinate intermediate [Co(N2S2)(CNtBu)2](Et4N) complex 5 was readily oxidized to sulfinates with a stoichiometric amount of this oxidant. The resulting complex 6 crystallizes with an octahedral structure. The SO bonds of the SO2 groups are almost equivalent (approximately 1.483 and approximately 1.453 A). The isonitrile is linearly bonded to the cobalt with a Co-C-N angle of 177.5 degrees and a very short C-N(tBu) distance of 1.13 A, which has a triple bond character. As expected for six-coordinate CoIII complexes, 5 and 6 are diamagnetic in agreement with their 1H and 13C NMR spectra. The SO2 IR bands are located at 1210 cm(-1) (v(as)SO2) and 1070 cm(-1) (v(s)SO2), while the CN vibration of the isonitrile is observed at 2170 cm(-1) in 5 and 2210 cm(-1) in 6. Very recently, it has been reported in the literature that oxidation of the coordinated thiolates was required for activity of both Fe and Co nitrile hydratases. Complex 6, with two oxidized thiolates trans to two deprotonated carboxamido nitrogens, is the first to have an in-plane closely related to that of the Co-NHase active site.     

Kinetic resolution of ligand binding to the alpha and beta chains within human hemoglobin.

Nitric oxide has been used as a chain-specific, spin label of unliganded heme groups present in kinetic mixtures of human hemoglobin and n-butyl isocyanide. In these experiments, deoxyhemoglobin was reacted with n-butyl isocyanide for a controlled time and then mixed rapidly with a high concentration of nitric oxide to fill residual, unoccupied heme sites. The final mixture was frozen immediately after formation to prevent any displacement of bound isonitrile. The EPR spectrum of the frozen sample was resolved into alpha and beta nitric oxide components; these reflect the relative proportions of alpha- and beta-heme sites which were unoccupied by n-butyl isocyanide. Individual time courses for the alpha and beta subunits were obtained by varying the time between the formation of the isonitrile/hemoglobin mixture and its reaction with nitric oxide. At pH 7.0 only the beta chain time course exhibits an initial rapid phase; the alpha chain time course is monophasic, exhibiting almost, exponential behavior. This result shows unequivocally that the beta-hemes within deoxyhemoglobin react much more rapidly with n-butyl isocyanide than the alpha hemes.     

Making M–CN bonds from M–Cl in (PONOP)M and (dippe)Ni systems (M = Ni, Pd, and Pt) using t-BuNC

C–N bond activation of tert-butyl isocyanide in methanol using 2,6-bis(di-tert-butylphosphinito)pyridine (PONOP) metal (Ni, Pd, Pt) complexes and (dippe)NiCl₂ are reported. t-BuOMe and t-BuCl were detected as organic products by GC–MS. Substitution of the metal-chloride by one molecule of tert-butyl isocyanide followed by carbonium ion loss/nucleophilic attack by chloride anion or methanol led to formation of a metal-cyanide bond.     

Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several site-directed mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å. We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys¹⁰¹) that is required for catalysis and interacts with Asp¹⁷, Thr¹⁰², and an ordered water molecule in the active site. Asp¹⁷ has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys¹⁰¹ and Asp¹⁷, Thr¹⁰² is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 Å C-α root mean square deviation), structural differences in the vicinity of Cys¹⁰¹ disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue.     

Isonitrile derivatives of polysaccharides as supports for the covalent fixation of proteins and other ligands.

A method for the introduction of side chains containing isonitrile (isocyanide, functional group) on the backbone of polysaccharides and other hydroxylic polymers was developed. The method was based on (a) ionization of some of the hydroxyl groups on the polymer by treatment with a strong base (tert-butoxide) in a polar aprotic solvent (dimethylsulfoxide), and (b) introduction of side chains containing isonitrile groups by nucleophilic attack of the polymeric alkoxide ions on a low molecular weight isonitrile containing a good leaving group in the omega-position, (1-tosyl-3-isocyanopropane). By this method, the side chains containing the-NC functional groups are attached to the polymeric backbone via stable ether bonds. The isonitrile derivatives of cellulose, linear and cross-linked dextran and cross-linked agarose utilized for the covalent fixation of high and low molecular weight ligands by four-component reactions carried out in aqueous medium, at neutral pH.     

Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase

Purified polymorphonuclear leukocyte elastase degraded native human liver type III collagen at 27 degrees C by making a cleavage through the triple helix. The enzyme had no effect on human type I collagen. The reaction was inhibited by phenylmethanesulfonyl fluoride (PhCH2SO2F) but not by EDTA. The collagen reaction products were identical with those generated by human rheumatoid synovial collagenase when analyzed by polyacrylamide gel electrophoresis and gel filtration. NH2-trminal sequence analysis indicated that the enzyme cleaved at an isoleucyl-threonyl bond located 4 residues on the carboxyl side of the established cleavage site for animal collagenases. Therefore, it is likely that in pathologic states, type III collagen can be selectively depleted from the matrix by this enzyme.     

Asymmetrical nitrido tc-99m heterocomplexes as potential imaging agents for benzodiazepine receptors

The design, synthesis, and biological evaluation of nitrido technetium-99m complexes for imaging benzodiazepine receptors are described. The design was performed by selecting the precursor biologically active substrate desmethyldiazepam, and the reactive metal-containing fragment [(99m)Tc(N)(PXP)](2+) (PXP = diphosphine ligand) as molecular building-blocks for assembling the structure of the final radiopharmaceuticals through the application of the so-called 'bifunctional' and 'integrated' approaches. This required the synthesis of the ligands H(2)BZ1, H(2)C1, and H(2)C2 (Figures 1 and 2) derived from desmethyldiazepam. In turn, these ligands were reacted with [(99m)Tc(N)(PXP)](2+) to afford the complexes [(99m)Tc(N)(PXP)(L)] (L = BZ1, C1, C2). The chemical nature of the resulting Tc-99m radiopharmaceuticals was investigated using chromatographic methods, and by comparison with the analogous complexes prepared with the long-lived isotope Tc-99g and characterized by spectroscopic and analytical methods. Results showed that the complexes [(99m)Tc(N)(PXP)(L)] are neutral and possess an asymmetrical five-coordinated structure in which two different bidentate ligands, PXP and L, are coordinated to the same Tc[triple bond]N core. With the ligand H(2)BZ1, two isomers were obtained depending on the syn or anti orientation of the pendant benzodiazepine group relative to the Tc[triple bond]N multiple bond. Biodistribution studies of Tc-99m complexes were carried out in rats, and affinity for benzodiazepine receptors was assessed through in vitro binding experiments on isolated rat's cerebral membranes using the corresponding Tc-99g complexes.     

Structural study of anhydrous tendon chitosan obtained via chitosan/acetic acid complex

The molecular structure and packing arrangement of anhydrous tendon chitosan was determined by the X-ray fibre diffraction method together with the linked-atom least-squares refinement technique. The specimen was prepared from chitosan/acetic acid complex which was obtained by exposing tendon chitosan to acetic acid vapour at room temperature for several days. There is high degree of orientation and crystallinity compared with the specimen obtained by the annealing method. Two chitosan chains are present in an orthorhombic unit cell of dimensions a = 8.26(2), b = 8.50(1), c (fibre axis) = 10.43(2) A and space group P2(1)2(1)2(1). The 2-fold helical chain is stabilised by O3 triple bond O5 hydrogen bond with the gt orientation of O6. There are direct hydrogen bonds (N2 triple bond O6) between adjacent chains along the a-axis, which makes a sheet structure parallel to the ac-plane. On the other hand, no hydrogen bond is found between the sheets.     

A comparison of the geminate recombination kinetics of several monomeric heme proteins

The geminate rate constants for CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide rebinding to soybean leghemoglobin and monomeric component II of Glycera dibranchiata hemoglobin were measured at pH 7, 20 degrees C using a dye laser with a 30-ns square-wave pulse. The results were compared to the corresponding parameters for sperm whale myoglobin and the isolated alpha and beta subunits of human hemoglobin (Olson, J.S., Rohlfs, R.J., and Gibson, Q.H. (1987) J. Biol. Chem., 262, 12930-12938). The rate-limiting step for O2, NO, and isonitrile binding to all five proteins is ligand migration up to the initial geminate state, and the rate of this process determines the overall bimolecular association rate constant for these ligands. In contrast, iron-ligand bond formation limits the overall bimolecular rate for CO binding. The distal pockets in leghemoglobin and in Glycera HbII are approximately 10 times more accessible kinetically to diatomic ligands than that in sperm whale myoglobin. This difference accounts for the much larger association rate constants (1-2 x 10(8) M-1 s-1) that are observed for O2 and NO binding to leghemoglobin and Glycera HbII. The rates of isonitrile migration through leghemoglobin are also very large and indicate a very fluid or open distal structure near the sixth coordination position. In contrast, there is a marked decrease in the rate of migration up to and away from the sixth coordination position in Glycera HbII with increasing ligand size. These results were also used to interpret previously published rate constants and quantum yields for the high (R) and low (T) affinity states of human hemoglobin. In contrast to the differences between the monomeric proteins, the differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.     

Novel procedures for preparing 99mTc(III) complexes with tetradentate/monodentate coordination of varying lipophilicity and adaptation to 188Re analogues

Improved methods are presented for the preparation of 99mTc and 188Re mixed-ligand complexes with tetradentate and monodentate ligands of the general formula [MIII(Lm)(Ln)] (M = Tc, Re; Lm = NS3 or NS3COOH; Ln = isocyanide or phosphine). To avoid the undesired formation of reduced-hydrolyzed species of both metals, the preparation of complexes is performed in a two-step procedure. At first the Tc(III)- or Re(III)-EDTA complex is formed which reacts in a second step with the tripodal ligand 2,2',2' '-nitrilotris(ethanethiol) (NS3) or its carboxyl derivative NS3COOH (a) and the monodentate phosphine ligands (triphenylphosphine L1, dimethylphenylphosphine L2) or isocyanides (tert-butyl isonitrile L3, methoxyisobutyl isonitrile L4, 4-isocyanomethylbenzoic acid-L-arginine L5, 4-isocyanomethylbenzoic acid-L-arginyl-L-arginine L6, 4-isocyanomethylbenzoic acid-neurotensin(8-13) L7) to the so-called '4+1' complex. Copper(I) isocyanide complexes are used for preparing the '4+1' complexes. That facilitates storage stability and allows kit formulations, and, moreover, enables the formation of 188Re complexes in acidic solution. Only micromolar amounts of the monodentate ligand are needed, and that results in high specific activity labeling of interesting molecules. The lipophilicity of complexes can be controlled by introducing a carboxyl group into the tetradentate ligand and/or derivatization of the monodentate ligands. Furthermore, the carboxyl group enables the conjugation of biomolecules. As an example, the neurotensin derivative CN-NT(8-13) was prepared and labeled with 99mTc according to the '4+1' approach, and its behavior in vivo was studied.     

Characterization of the functional role of Asp141, Asp194, and Asp464 residues in the Mn2+-L-malate binding of pigeon liver malic enzyme

Pigeon liver malic enzyme was inactivated and cleaved at Asp141, Asp194, and Asp464 by the Cu2+-ascorbate system in acidic environment. Site-specific mutagenesis was performed at these putative metal-binding sites. Three point mutants, D141N, D194N, and D464N; three double mutants, D(141,194)N, D(194,464)N, and D(141,464)N; and a triple mutant, D(141,194,464)N; as well as the wild-type malic enzyme (WT) were successfully cloned and expressed in Escherichia coli cells. All recombinant enzymes, except the triple mutant, were purified to apparent homogeneity by successive Q-Sepharose and adenosine-2',5'-bisphosphate-agarose columns. The mutants showed similar apparent Km,NADP values to that of the WT. The Km,Mal value was increased in the D141N and D194N mutants. The Km,Mn value, on the other hand, was increased only in the D141N mutant by 14-fold, corresponding to approximately 1.6 kcal/mol for the Asp141-Mn2+ binding energy. Substrate inhibition by L-malate was only observed in WT, D464N, and D(141,464)N. Initial velocity experiments were performed to derive the various kinetic parameters. The possible interactions between Asp141, Asp194, and Asp464 were analyzed by the double-mutation cycles and triple-mutation box. There are synergistic weakening interactions between Asp141 and Asp194 in the metal binding that impel the D(141,194)N double mutant to an overall specificity constant [k(cat)/(Kd,Mn Km,Mal Km,NADP)] at least four orders of magnitude smaller than the WT value. This difference corresponds to an increase of 6.38 kcal/mol energy barrier for the catalytic efficiency. Mutation at Asp464, on the other hand, has partial additivity on the mutations at Asp141 and Asp194. The overall specificity constants for the double mutants D(194,464)N and D(141,464)N or the triple mutant D(141,194,464)N were decreased by only 10- to 100-fold compared to the WT. These results strongly suggest the involvement of Asp141 in the Mn2+-L-malate binding for the pigeon liver malic enzyme. The Asp194 and Asp464, which may be oxidized by nonspecific binding of Cu2+, are involved in the Mn2+-L-malate binding or catalysis indirectly by modulating the binding affinity of Asp141 with the Mn2+.     

Isocyanide binding to the copper(I) centers of the catalytic core of peptidylglycine monooxygenase (PHMcc)

Binding of the Cu(I)-specific ligands 2,6-dimethylphenyl isocyanide (DIMPI) and isopropyl isocyanide (IPI) to the reduced form of peptidylglycine monooxygenase (PHM) is reported. Both ligands bind to the methionine-containing CuM center, eliciting FTIR bands at 2,138 and 2,174 cm(-1), respectively, but appear unable to coordinate at the histidine-containing CuH center in the wild-type enzyme. This chemistry parallels that previously observed for CO binding to the reduced PHM catalytic core (PHMcc). However, in contrast to the CO chemistry, peptide substrate binding did not induce binding of the isocyanide at CuH. XAS confirmed the binding of DIMPI at CuM via the observation of a short Cu-C interaction at 1.87 A and by the lengthening of the Cu-S(methionine) bond length by 0.06 A. Similarly, FTIR studies on DIMPI binding to the M314I and H172A mutant forms of reduced PHMcc confirmed the assignment of the 2,138-cm(-1) IR band as a CuM-DIMPI complex, but surprisingly also showed DIMPI binding to CuH, as indicated by a band at 2,148 cm(-1). An inorganic complex, [Cu(1,2-Me2Im)2(DIMPI)](PF6), was synthesized and its crystal structure was determined as a model for the interaction of isocyanides with imidazole-containing Cu(I) complexes. Comparison of EXAFS data for the protein and model suggests that DIMPI probably binds to CuM in a tilted fashion, similar to that of ethyl isocyanide binding to myoglobin.     

Role of hydrogen bond networks and dynamics in positive and negative cooperative stabilization of a protein

Cooperativity mediated through hydrogen bond networks in yeast iso-1-cytochrome c was studied using a thermodynamic triple mutant cycle. Three known stabilizing mutations, Asn 26 to His, Asn 52 to Ile, and Tyr 67 to Phe, were used to construct the triple mutant cycle. The side chain of His 26, a wild-type residue, forms two hydrogen bonds that bridge two substructures of the wild-type protein, and Tyr 67 and Asn 52 are part of an extensive buried hydrogen bond network. The stabilities of all variants in the triple mutant cycle were determined by guanidine hydrochloride denaturation methods and used to determine the pairwise, Delta(2)G(int), and triple interaction energies. His 26 and Ile 52 interact cooperatively (Delta(2)G(int) is 1-2 kcal/mol), whereas the two other pairs of mutations interact anticooperatively (Delta(2)G(int) is -0.5 to -1.5 kcal/mol). Previously reported structural data for iso-1-cytochrome c variants containing these mutations show that changes in the strength of the His 26 to Glu 44 hydrogen bond, apparently caused by changes in main chain dynamics, provide a mechanism for the long distance (His 26 to Phe 67 and His 26 to Ile 52) propagation of pairwise interaction energies. Opposing changes in the strength of the His 26 to Glu 44 hydrogen bond caused by the N52I and Y67F mutations generate a negative triple interaction energy (-0.9 +/-0.7 kcal/mol) that combined with cancellation of cooperative and anticooperative pairwise interactions produce apparent additivity for the stabilizing effects of the single mutations in the triple mutant variant.     

Function analysis of conserved amino acid residues in a Mn2+-dependent protein phosphatase, Pph3, from Myxococcus xanthus

The Myxococcus xanthus protein phosphatase Pph3 belongs to the Mg(2+)- or Mn(2+)-dependent protein phosphatase (PPM) family. Bacterial PPMs contain three divalent metal ions and a flap subdomain. Putative metal- or phosphate-ion binding site-specific mutations drastically reduced enzymatic activity. Pph3 contains a cyclic nucleotide monophosphate (cNMP)-binding domain in the C-terminal region, and it requires 2-mercaptoethanol for phosphatase activity; however, the C-terminal deletion mutant showed high activity in the absence of 2-mercaptoethanol. The phosphatase activity of the wild-type enzyme was higher in the presence of cAMP than in the absence of cAMP, whereas a triple mutant of the cNMP-binding domain showed slightly lower activities than those of wild-type, without addition of cAMP. In addition, mutational disruption of a disulphide bond in the wild-type enzyme increased the phosphatase activity in the absence of 2-mercaptoethanol, but not in the C-terminal deletion mutant. These results suggested that the presence of the C-terminal region may lead to the formation of the disulphide bond in the catalytic domain, and that disulphide bond cleavage of Pph3 by 2-mercaptoethanol may occur more easily with cAMP bound than with no cAMP bound.     

The synthesis and cyclic voltammetry of select ferrocene piano-stool isocyanide complexes

Ferrocene piano-stool isocyanide complexes ([CpFeL₃]⁺, Cp = η⁵-C₅H₅, L = tert-butyl isocyanide (1), cyclohexyl isocyanide (2), and 2,6-dimethylphenyl isocyanide (3)) are formed by chemical oxidation of ferrocene in the presence of a stoichiometric amount of isocyanide ligand (1:3). The complexes are characterized by elemental analysis, routine spectroscopic methods (IR, ¹H NMR, ¹³C NMR, and UV-Vis), and cyclic voltammetry.     

Alkyne substrate interaction within the nitrogenase MoFe protein

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC triple bond CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein alpha-70(Val) residue, whose side chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of alpha-70(Val) by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC triple bond C-CH(3)), whereas, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the alpha-70(Val) residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC triple bond C-CH(2)-CH(3)). This protein shows no detectable reduction of the internal alkyne 2-butyne (H(3)C-C triple bond C-CH(3)). In contrast, substitution of the nearby alpha-191(Gln) residue by alanine, in combination with the alpha-70(Ala) substitution, does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein.     

A novel approach to the high-specific-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP = diphosphine ligand).

A new labeling approach for incorporating bioactive peptides into a technetium-99m coordination complex is described. This method exploits the chemical properties of the novel metal-nitrido fragment [99mTc(N)(PXP)]2+, composed of a terminal Tc[triple bond] N multiple bond bound to an ancillary diphosphine ligand (PXP). It will be shown that this basic, molecular building block easily forms in solution as the dichloride derivative [99mTc(N)(PXP)Cl2], and that this latter complex selectively reacts with monoanionic and dianionic, bidentate ligands (YZ) having soft, pi-donor coordinating atoms to afford asymmetrical nitrido heterocomplexes of the type [99mTc(N)(PXP)(YZ)]0/+ without removal of the basic motif [99mTc(N)(PXP)]2+. The reactions of the amino acid cysteine was studied in detail. It was found that cysteine readily coordinates to the metal fragment [99mTc(N)(PXP)]2+ either through the [NH2, S-] pair of donor atoms or, alternatively, through the [O-, S-] pair, to yield the corresponding asymmetrical complexes in very high specific activity. Thus, these results were conveniently employed to devise a new, efficient procedure for labeling short peptide sequences having a terminal cysteine group available for coordination to the [99mTc(N)(PXP)]2+ fragment. Examples of the application of this novel approach to the labeling of the short peptide ligand H-Arg-Gly-Asp-Cys-OH (H(2)1) and of the peptidomimetic derivative H-Cys-Val-2-Nal-Met-OH (H2) will be discussed.     

Steric and hydrophobic effects in alkyl isocyanide binding to Rhodospirillum molischianum cytochrome c'

Equilibrium constants for the binding of a series of alkyl isocyanides to ferrous cytochrome c' from Rhodospirillum molischianum have been measured spectrophotometrically. The equilibrium constants range from 3.3 M-1 to 2.6 x 10(2) M-1 and follow the order methyl greater than ethyl less than n-propyl less than tert-butyl less than n-butyl less than amyl less than cyclohexyl less than n-hexyl. The decrease in equilibrium constant from methyl to ethyl isocyanide provides evidence for a steric interaction between the ligand and the protein. The increase in equilibrium constant from ethyl to n-hexyl isocyanide is accounted for by a favorable partitioning of the ligand into a hydrophobic heme coordination site. The effect of steric interactions on the differences in the binding constants has been further evaluated by comparing the alkyl isocyanide and CO binding constants for the ferrous cytochrome c' to those of a sterically unconstrained model heme complex in a detergent micelle. The results indicate that the heme coordination site of the ferrous cytochrome c' is severely sterically hindered, similar to that of the reported crystal structure of Rs. molischianum ferric cytochrome c'.