Electrochemical cells for voltammetry, coulometry, and protein activity assays of small-volume biological samples.

Cell designs, experimental protocols, and results for electrochemical investigation of small quantitites of biological materials under anaerobic conditions are reported. Three types of electrochemical experiments are considered: (i) cyclic voltammetry of 20- to 100-microliters samples; (ii) direct coulometry of 0.5- to 1.5-ml samples; and (iii) an electrochemically initiated protein activity assay which includes provision for analysis of gaseous reaction products and correlation with electron flux. The first two procedures are illustrated by measurement of the formal electrode potential (E0') and number of electrons transferred (n) in redox reactions of small quantities of biological and inorganic materials. The third procedure is illustrated by assaying the activity of the MoFe protein plus Fe protein complex from Azotobacter vinelandii nitrogenase for reduction of C2H2 to C2H4.

Purification and spectroscopic characteristics in N-methylformamide of the Azotobacter vinelandii Fe-Mo cofactor.

The iron-molybdenum cofactor from Azotobacter vinelandii can be removed from significant amounts of extraneous iron and other contaminants using anaerobic gel filtration. Electronic absorption spectra of the so-purified FeMoco along with analysis of the so-called 'easily complexed' iron are suggestive that FeMoco occupies at least two different states in N-methylformamide solution. Batch-related differences in spectral characteristics of independently isolated FeMoco samples are demonstrated. Non-cofactor iron, found in unpurified FeMoco, may affect the interpretation of ligand binding and other experiments probing FeMoco structure and reactivity. Oxidized FeMoco is shown to be clearly discernable from the semi-reduced species by means of electronic spectroscopy, and this method now forms a convenient analytical tool for study of the chemistry and electronic structure of FeMoco.

Mössbauer spectroscopy applied to the oxidized and semi-reduced states of the iron-molybdenum cofactor of nitrogenase.

Mössbauer parameters at 125K for both the oxidized and semi-reduced states of FeMoco isolated from the MoFe protein of Azotobacter vinelandii nitrogenase of delta/Fe = 0.32 and 0.37 mm/s and delta Eq = 0.84 and 0.71 mm/s, respectively, are reported. FeMoco(ox) fits the Debye model perfectly from 4.2-125K and has a S = 0 ground state. FeMoco(ox) apparently contains 10-20% FeMoco(s-r) and vice versa, possibly as a result of the spontaneous oxidation phenomenon. Quantitation of the spectra indicates a Fe:Mo ratio of 5 +/- 1:1 and the similar quadrupole splittings and isomer shifts suggest a similar environment for all iron atoms.

Isolated iron-molybdenum cofactor of nitrogenase exists in multiple forms in its oxidized and semi-reduced states.

Electrochemical and EPR spectroscopic experiments demonstrate that the isolated iron-molybdenum cofactor from the molybdenum-iron protein of nitrogenase from Azotobacter vinelandii exists in multiple forms in both its oxidized and semi-reduced states. The particular forms found in either oxidation state appear to be a function of the acid/base status of the solvent, N-methylformamide. In "alkaline" N-methylformamide, a single, detectable form of iron-molybdenum cofactor is observed for both oxidized and semi-reduced states. The semi-reduced form, termed R(s-r), is the one previously recognized with an S = 3/2 EPR spectrum with apparent g values of 4.6, 3.4, 2.0. Its oxidized counterpart, termed B(ox), is characterized electrochemically by a differential pulse voltammetric reduction peak at -0.37 V versus the normal hydrogen electrode. In "acidic" solvent, two distinct, previously unrecognized redox pairs of iron-molybdenum cofactor forms exist. The two semi-reduced forms, N(s-r) and W(s-r), are characterized by EPR spectra with g = 4.5, 3.6, 2.0 and g = 4.9, 3.1, 1.9, respectively. Their oxidized counterparts, A(ox) and C(ox), have differential pulse voltammetric reduction peaks at -0.32 and -0.43 V versus the normal hydrogen electrode, respectively. Manipulations of either the isolation protocol or the sample conditions affects both the type and distribution of forms present. Each form likely corresponds to a biologically significant state of the cofactor cluster within the protein.

Iron-molybdenum cofactor of nitrogenase: electrochemical determination of the electron stoichiometry of the oxidized/semi-reduced couple.

The number of electrons involved in the more positive of the two redox couples of the iron-molybdenum cofactor of Azotobacter vinelandii nitrogenase has been investigated by controlled potential coulometry in both the oxidizing and reducing directions. A n value of 1 was determined for interconversion of the oxidized and semi-reduced states of the cofactor. This electron count was confirmed by double integration of the S = 3/2 electron paramagnetic resonance signal exhibited by the semi-reduced state.

Elicitation of thiomolybdates from the iron-molybdenum cofactor of nitrogenase. Comparison with synthetic Fe-Mo-S complexes.

Aerial oxidation of the iron-molybdenum cofactor (FeMoco) of Azotobacter vinelandii nitrogenase has been shown to yield either the tetrathiomolybdate ion ([MoS4]2-) or the oxotrithiomolybdate ion ([MoOS3]2-), depending on the reaction conditions. Thus, when N-methylformamide (NMF) solutions of FeMoco either were titrated with measured aliquots of air or were diluted with air-saturated NMF, [MoOS3]2- was found to be the predominant product while dilution of NMF solutions of FeMoco with air-saturated methanol produced [MoS4]2- almost exclusively. Similar aerial oxidation of solutions of chemically synthesized Fe-Mo-S clusters showed that significant information about the molybdenum environment in these species could be deduced from the nature of the elicited thiomolybdates. The differences in decomposition products as a function of solvent are postulated to be due to the loss through precipitation of the reducing agent sodium dithionite on addition of methanol but not NMF. These overall decomposition results are discussed in the context of recent X-ray absorption spectroscopic data which suggest the presence of an 'MoS3' core in FeMoco. A possible mechanism whereby [MoS4]2- might be rapidly formed from this core is presented.