Detection of volatile organic compounds using porphyrin derivatives.

Seven different porphyrin compounds have been investigated as colorimetric gas sensors for a wide range of volatile organic compounds. The porphyrins examined were the free base and Mg, Sn, Zn, Au, Co, and Mn derivatives of 5,10,15,20-tetrakis[3,4-bis(2-ethylhexyloxy)phenyl]-21H,23H-porphine. Chloroform solutions of these materials were prepared and changes in their absorption spectra induced by exposure to various organic compounds measured. The porphyrins that showed strong responses in solution were selected, and Langmuir-Blodgett films were prepared and exposed to the corresponding analytes. This was done to determine whether they are useful materials for solid state thin film colorimetric vapor sensors. Porphyrins that readily coordinate extra ligands are shown to be suitable materials for colorimetric volatile organic compound detectors. However, porphyrins that already have bound axial ligands when synthesized only show a sensor response to those analytes that can substitute these axial ligands. The Co porphyrin displays a considerably larger response than the other porphyrins investigated which is attributed to a switch between Co(II) and Co(III) resulting in a large spectral change.

Alkylamine sensing using langmuir-blodgett films of n-alkyl-N-phenylamide-substituted zinc porphyrins.

Two porphyrin compounds, zinc(II) 5,10,15,20-tetrakis(3,5,5-trimethyl- N-phenylhexanamide)porphyrin and zinc(II) 5,10,15,20-tetrakis(2,2-dimethyl- N-phenylpropanamide)porphyrin, have been investigated as possible candidates for the detection of alkylamines. UV-visible spectroscopy has shown that their solution absorption spectra are significantly modified upon interaction with a range of organic analytes, including acetic acid, butanone, ethylacetate, hexanethiol, octanal, octanol, alkylamines, and trimethylphosphite. Large spectral changes are observed for the family of alkylamines as a result of the specific affinity between zinc and the amine moiety. Langmuir-Blodgett (LB) films of the porphyrins have been fabricated in order to assess their solid-state sensing capability toward amines. The surface pressure-area (Pi- A) isotherms reveal a clear three-phase Langmuir film behavior and show that these monolayer films may be compressed to a relatively high surface pressure ( approximately 40-50 mN m (-1)). The isotherm data alongside molecular modeling suggest a relatively flat orientation of the porphyrin rings of both compounds: that is, a mutually parallel alignment of the plane of the porphyrin ring and that of the water surface. LB films deposited at 15 mN m (-1) have been exposed to alkylamine vapor (carried by N 2). A red shift and increase in intensity of the Soret band absorbance is observed which can be reversed by flowing pure N 2 over the gently heated sample (60 degrees C) after exposure. Primary amines were expected to invoke the greatest sensing response due to (i) their larger association constants with these porphyrins compared to secondary and tertiary amines and (ii) the ease of diffusion of amines which is expected to follow the order primary > secondary > tertiary due to the steric hindrance arising from the bulky secondary and tertiary amines. However, the magnitude of the absorbance change is largest for exposure to the secondary amines, dipropylamine and dibutylamine, for both porphyrins, compared to primary and tertiary amines. This trend follows that observed when the amines were added to solutions of the porphyrins. The rate of response of the porphyrin LB films falls as the molecular weight of the diffusing alkylamine increases. Furthermore, a greater rate of response is observed for the phenylhexanamide porphyrin compared to the phenylpropanamide porphyrin due to its lower molecular density within the LB film and therefore more porous structure.

Development and optimization of porphyrin gas sensing LB films.

A series of meso-substituted metal-free porphyrins has been developed which show high sensitivity to NO2 gas in the sub-5 ppm concentration range. By selecting different substituents, it has been possible to improve in a systematic manner the response time and sensitivity of the porphyrin LB film to NO2. Initially, a sulphonamino substituent yielded a fairly long response time of around 450 s but this was shortened considerably when this substituent was changed for a stearamido group. Further modifications resulted in achieving a porphyrin LB film which exhibited a t50 response time of only 11 s. By using an optically inert calixarene host material in which the porphyrin guest was incorporated, it was possible to obtain t50 values as low as 5 s.

Pyroelectric and conduction properties of Z-type calix[4] acid Langmuir-Blodgett films.

Non-centrosymmetric Z-type Langmuir-Blodgett (LB) films were prepared by transferring a calix[4] acid monolayer from a subphase of Millipore water (18 Momegacm(-1)) onto aluminised glass substrates. Electrical measurements were performed at room temperature on LB films with a sandwich structure comprising a 50 nm thick thermally evaporated aluminium film. A pyroelectric figure of merit of 2.23 microC m(-2)K(-1) is reported for this LB system. The low voltage value of conductivity is 1.82 x 10(-12) Sm(-1). The electrode-limited Schottky effect is responsible for the conduction mechanism at a relatively high field due to the dc bias and the barrier potential height is determined to be 1.72 eV. The ac conductance for both samples shows a typical power law dependence with a value of approximately 0.86 for the exponent.

Identification of amino acid substitutions that confer a high affinity for sulfaphenazole binding and a high catalytic efficiency for warfarin metabolism to P450 2C19.

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacement of amino acids 283-338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a KS < 10 microM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (KS < 5 microM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.

Heterologous expression of human drug-metabolizing enzymes.

This article is a report on a symposium held at the March 1997 meeting of the American Society for Pharmacology and Experimental Therapeutics in San Diego. Current developments in the heterologous expression of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione transferase, and UDP-glucuronosyltransferase enzymes are described. Systems include bacteria, insect cells, and transient and stable mammalian cells. Uses of the products are described for discernment of which enzymes are involved in metabolism of drugs, genotoxicity assays, mutagenesis (for structure-activity relationships), large scale production of enzyme products, antibody production, and production of proteins for biophysical studies.

Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus.

A hydrophobic segment present in the N-terminus of microsomal P450s is thought to serve as a membrane anchor. A variant of P450 2C3 was constructed, P450 2C3d, that lacked the putative membrane-spanning segment of the N-terminus, residues 3-20. This construct also incorporated substitutions of an alanine for 2Asp to facilitate expression in Escherichia coli and of serines for 24His and 25Gly to introduce a restriction site. P450 2C3d is expressed at relatively high levels in E. coli, 800-1200 nmol/liter of culture medium. In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression in E. coli, the subcellular distribution of P450 2C3d in E. coli is dependent on the ionic strength of the buffer used for cell disruption. In low ionic strength buffers, 2C3d was mainly localized in the membrane fraction, whereas in buffers containing 1 M NaCl or 0.5 M KPi, P450 2C3d was predominantly found in the soluble fraction, indicating that deletion of the hydrophobic segment converted the intrinsic membrane protein to an extrinsic one. P450 2C3d was further modified by the incorporation of four histidine residues at the C-terminus (P450 2C3dH), and this enzyme could be purified in the absence of detergent using immobilized metal affinity chromatography following extraction from isolated membranes in high salt buffers. The catalytic properties of the purified, modified enzymes are similar to those of the native enzyme. Size-exclusion chromatography indicated that 2C3dH and 2C3d are predominantly dimers, whereas 2C3 is a larger oligomer (> 8-mer). Moreover, the detergents sodium cholate and Chaps each dissociate the dimers of 2C3dH to monomers at concentrations that do not alter the aggregation state of 2C3. These modifications are likely to facilitate attempts to crystallize the catalytic domains of microsomal P450s.

Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51.

The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6. Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18. The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation. Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards. Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected.

Diazepam metabolism by cDNA-expressed human 2C P450s: identification of P4502C18 and P4502C19 as low K(M) diazepam N-demethylases.

The present study provides a detailed kinetic analysis of diazepam metabolism by all four known members of the human P4502C subfamily expressed from their cDNAs in Escherichia coli. Both P4502C18 and P4502C19 were found to be low K(M) diazepam N-demethylases with apparent K(M) values of 24 +/- 4 microM and 21 +/- 3 microM, respectively. These values closely resemble the low K(M) component of diazepam N-demethylase activity exhibited by human liver microsomes. In addition, P4502C19 also catalyzed diazepam 3-hydroxylation with a K(M) value of 21 +/- 9 microM. Although P4502C8 was essentially inactive in catalyzing diazepam metabolism, P4502C9 catalyzed the N-demethylation with a relatively high K(M) of 80 +/- 15 microM and an overall 3- to 6-fold lower catalytic efficiency, compared with P4502C18 and P4502C19, respectively. At a substrate concentration of 10 microM, diazepam N-demethylation in a panel of human liver microsomes was inhibited 42 +/- 12% (mean +/- SD, N = 6) by a polyclonal anti-CYP2C antibody. In the same experiment, 3-hydroxylation remained unaffected (<10% inhibition). 1 microM of the CYP3A inhibitor ketoconazole inhibited 37 +/- 19% of the N-demethylation and 86 +/- 5% of 3-hydroxylation. Estimates of relative contributions to diazepam N-demethylation of P4502C9 (8 +/- 4%), P4502C18 (<2%), and P4502C19 (33 +/- 14%) and to diazepam 3-hydroxylation of P4502C19 (9 +/- 3%) based on the kinetic parameters of the recombinant enzymes and on specific contents of the individual 2C P450s determined in immunoblots are consistent with the inhibition data. In conclusion, these data confirm that both P4502C19 and P4503A are major contributors to human liver microsomal diazepam N-demethylation at low substrate concentrations, whereas P4503A is the major enzyme responsible for 3-hydroxylation.

A universal approach to the expression of human and rabbit cytochrome P450s of the 2C subfamily in Escherichia coli.

Human cytochrome P450s 2C8, 2C9, 2C18, and 2C19 and rabbit cytochrome P450s 2C1, 2C2, 2C4, 2C5, and 2C16 were expressed from their respective cDNAs in Escherichia coli as chimeric enzymes in which a portion of the N-terminal membrane anchor sequence was replaced with a modified sequence derived from P450 17A. For 2C1 and 2C2 removal of the extraneous 3'-untranslated sequence allowed the successful expression of constructs that were unproductive in its presence. The levels of expression varied from 180 to 1500 nmol/liter of culture and the addition of delta-aminolevulinic acid to the culture media increased the amount of spectrally detectable P450 for several of these enzymes 2- to 10-fold. The catalytic properties of the modified human 2C P450s expressed in E. coli were concordant with previously published data for several marker substrates including (S)-mephenytoin for P450 2C19, tolbutamide and tetrahydrocannabinol (THC) for P450 2C9, and taxol for P450 2C8. Interestingly, P450 2C19 catalyzed the 21-hydroxylation of progesterone and, to a lesser extent, catalyzed the formation of 16 alpha-hydroxyprogesterone. The rabbit enzyme P450 2C16 catalyzed the formation of 17 alpha- and 16 alpha-hydroxyprogesterone in addition to 21-hydroxylation. P450 2C19 also catalyzed the methylhydroxylation of tolbutamide and the 7-hydroxylation of THC at rates that were similar to or greater than that of P450 2C9. This work has identified important factors required for the high-level expression of 2C subfamily P450s in E. coli. The availability of these enzymes will facilitate detailed kinetic measurements for known and yet to be identified substrates.

Inactivation of Escherichia coli-expressed rabbit cytochrome P-450 2C enzymes by 17 beta-substituted steroids.

The specific inactivation of rabbit cytochromes P-450 2C by 17 beta-substituted steroids has been investigated by using purified, Escherichia coli-expressed enzymes. The expressed P-450s provided a means to characterize accurately the effects of 21,21-dichloroprogesterone, 21,21-dichloropregnenolone, 21-chloro-21-fluoropregnenolone, pregn-5,20-diene-3 beta-ol and pregn-4,20-diene-3-one on progesterone hydroxylation by P-450 2C5, 2C4, 2C3 and 2C3v. Previous studies using rabbit liver microsomes had suggested that 21-chloro-21-fluoropregnenolone is a selective inactivator of 2C5, a progesterone 21-hydroxylase. Studies of the expressed P-450 2C forms showed little selectivity of 21,21-dichloroprogesterone, pregn-5,20-diene-3 beta-ol or pregn-4,20-diene-3-one, whereas 21,21-dichloropregnenolone and 21-chloro-21-fluoropregnenolone preferentially inactivate 2C5. The data indicate the importance of progesterone 21-hydroxylase activity in facilitating selective mechanism-based inactivation of 2C subfamily P-450s by 21,21-dihalogenated steroids. Studies of the inactivation of P-450 2C16, a progesterone 16 alpha-hydroxylase, by the three dihalogenated steroids yielded results consistent with previous findings of 16 alpha-hydroxylase inactivation in rabbit liver microsomes from the inbred B/J strain. Additionally, two mutants, 2C3v:V113A and 2C3v:V113A, T364N were created which confer progesterone 21-hydroxylation on 2C3v. The single mutant, a 6 beta- and 21-hydroxylase, is inactivated rapidly by all three of the 21,21-dihalogenated steroids, whereas the double mutant, a 16 alpha- and 21-hydroxylase, is preferentially inactivated by 21,21-dichloroprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of the regiospecificity of progesterone metabolism by the mutagenesis of two key amino acid residues in rabbit cytochrome P450 2C3v.

A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.

Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid omega-hydroxylase and the cognate enzyme expressed in Escherichia coli.

A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library. Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney. The enzyme was expressed from its cDNA in Escherichia coli. A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively. Little or no activity was detected toward prostaglandins A1 and E1.

Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.

Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.

Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene.

1. A polyclonal, monospecific antibody to a constitutive, diabetes-inducible and insulin-reversible cytochrome P-450 isozyme (RLM6) was used to screen a male rat liver cDNA library in lambda gt 11. Six clones harbouring the RLM6 cDNA insert were isolated initially from the expression library and three of these were further plaque-purified and sub-cloned. A 1.1 Kb cDNA insert, representing approximately 65% of the expected full length cDNA was characterized by restriction endonuclease mapping and sequenced by the dideoxy chain-termination method. Comparison of the nucleotide sequence of RLM6 cDNA to that of ethanol-inducible P4502E1 rat cDNA showed the two cDNAs to be identical, the RLM6 cDNA corresponding to nucleotides 310-1402 of the P4502E1 sequence. 2. RLM6 cDNA probe was used in Northern blot and RNA dot blot hybridization analysis to demonstrate that both streptozotocin-induced diabetes and fasting significantly elevated the steady-state level of RLM6 mRNA in male rat liver. Increased RLM6 mRNA level in the diabetic rat resulted in increased RLM6 apoprotein synthesis when polysomal RNA was used in a cell-free, protein-synthesizing system, indicating that the elevated RLM6 level observed in diabetic rats was correlated directly with the increased RLM6 mRNA concentration. 3. Daily insulin treatment of diabetic rats reversed the diabetes-dependent increase in RLM6 mRNA in a time-dependent manner, returning to control values after approximately 2 weeks of continuous insulin treatment. This insulin-dependent decrease of the RLM6 mRNA level was paralleled by a similar time-dependent decrease in serum acetone concentration. 4. Treatment of the male diabetic rat with testosterone also resulted in a decrease in both RLM6 mRNA and in vitro translated apoprotein. 5. Modulation of RLM6 mRNA level in the diabetic rat by insulin and testosterone, and the nucleotide sequence similarity with that of P4502E1 confirms that diabetes-inducible P450RLM6 and ethanol-inducible P4502E1 are coded for by the same gene.

The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein.

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.

The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein.

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein X ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.