Hydrogenase preparation

Affinity SFE cartridges are coming on the market for antibody preparation (protein G and protein A columns), for hydrogenase preparations (blue, red, and green dye columns), and activated cartridges that can bind proteins and amino-containing compounds to prepare affinity columns for specific uses are available. These may go beyond the basic definition of the SFE, but they are just simply variations on the same theme. 

Although the spectra of oxidized bacterial and plant ferredoxins are different, Fig. 4 shows that, when reduced, their absorption spectra are remarkably similar. In both cases, the absorbancy peaks in the visible region disappear, while there is little change in ultraviolet absorption. In Fig. 4 the ferredoxins were reduced by hydrogen gas with a bacterial hydrogenase preparation. When air was admitted into the cuvettes, both ferredoxins were auto-oxidized and showed their original absorption spectra (Fig. 3). Ferredoxins, therefore, are like cytochrome c in that reduction and oxidation is a reversible process. However, unlike the cytochromes, the ferredoxins are colored in the oxidized state and colorless when reduced. 

Racker 56) demonstrated the synthesis of carbohydrates from carbon dioxide and hydrogen in a cell-free system by bringing together many of the enzymes listed in Table IV. A spinach fraction furnished the phospho-pentokinase, carboxydismutase, phosphopentosisomerase, transketolase, transaldolase, and hexose diphosphatase. To this fraction were added the other enzymes, DPN+, ATP, and a hydrogenase preparation. The hydro-genase enzyme furnished DPNH in the presence of hydrogen. When this mixture was incubated at 25° for 60 minutes, the synthesis of fructose 6-phosphate could be demonstrated. 

Nickel-selenium coordination compounds have received attention in recent years, because a unique Ni-selenocysteine interaction was revealed in the active site of [FeNiSe]-hydrogenases.1083,1084 Of particular interest in this regard are mixed CO/selenolate complexes. Distorted square planar (393) was prepared from [CpNi(CO)]2, PhSeSePh, and [Fe(CO)3(SePh)3] and provides the first example of CO bound to a square planar Ni11 center in thiolate/selenolate environment.1085,1086 Upon addition of RSSR, species of the series [Ni(CO)(SR) (SePh)3 ] are formed. //(CO) ranges from 2,023 cm-1 to 2,043 cm-1 and is regarded as a spectroscopic reference for the CO binding site in [NiFeSe] hydrogenases. 

It has been known since the 19th century that hexacyanoferrates(II) react with concentrated sulphuric acid to give carbon monoxide the preparation of [Fe(CN)5(CO)]3- from these reagents was reported in 1913. It now seems likely that the conversion of coordinated -CN into -CO proceeds through -CONH2, here as in certain iron-containing hydrogenases (139). The stereospecific formation of/rac-[Fe(CN)3(CO)3]-then ds-[Fe(CN)4(CO)2]2- from [Fe(CO)4I2], as well as the stereospecific production of t vans - [Fe (CN), (CO )212 - from iron(II) chloride plus cyanide in an atmosphere of CO, appears to be under kinetic control, cis-[Fe(CN)4(CO)2]2- decomposes in minutes in aqueous solution, whereas... 

Six-fold averaged electron density map of the catalytic site of D. gigas [NiEe] hydrogenase in the as-prepared oxidised form at 0.25 nm resolution... 

A frequently asked question concerns the actual functional state of the crystallised protein. EPR analyses have shown, for example, that the enzyme present in aerobically prepared crystals of [NiFe] hydrogenase from D. gigas is mainly in its inactive, unready (Ni-A) state. However, it can be activated after prolonged incubation under hydrogen in the presence of methyl viologen (NiviHe et al. 1987). 

As described above, the combination of EPR and Mossbauer spectroscopies, when applied to carefully prepared parallel samples, enables a detailed characterization of all the redox states of the clusters present in the enzyme. Once the characteristic spectroscopic properties of each cluster are identified, the determination of their midpoint redox potentials is an easy task. Plots of relative amounts of each species (or some characteristic intensive property) as a function of the potential can be fitted to Nernst equations. In the case of the D. gigas hydrogenase it was determined that those midpoint redox potentials (at pFi 7.0) were —70 mV for the [3Fe-4S] [3Fe-4S]° and —290 and —340mV for each of the [4Fe-4S]> [4Fe-4S] transitions. 

Recently, the structure and spectral properties of a well-known diiron Fe model complex ( ji-SCH2CH2CH2S)Fe2(CO)6 were reinvestigated by Darensbourg and coworkers (Lyon et al. 1999). Also the Fe2(CO)4(CN)2 derivative was prepared and investigated. The v(CO) and v(CN) bands of the latter complex best fitted those found for the reduced D. vulgaris [Fe] hydrogenase and hence the possibility of an Fe -Fe pair in the reduced enzyme was suggested by the authors. 

The pharmaceutical and fine chemical industry might use pure hydrogenase or partially purified enzyme preparations in bioconversion applications such as regio and stereoselective hydrogenation of target compounds (van Berkel-Arts et al. 1986). Enzymes are able to catalyse such stereospecific syntheses with ease. However, the cofactors for the NAD-dependent oxidoreductases are expensive. The pyridine nucleotide-dependent hydrogenases such as those from Ralstonia eutropha and hyperthermophilic archaea (Rakhely et al. 1999) make it possible to exploit H2 as a low-cost reductant. The use of inverted micelles in hydrophobic solvents, in which H2 is soluble, has advantages in that the enzymes appear to be stabilized. 

Usually, activities of enzymes (hydrogenases included) are investigated in solutions with water as the solvent. However, enhancement of enzyme activity is sometimes described for non-aqueous or water-limiting surroundings, particular for hydrophobic (or oily) substrates. Ternary phase systems such as water-in-oil microemulsions are useful tools for investigations in this field. Microemulsions are prepared by dispersion of small amounts of water and surfactant in organic solvents. In these systems, small droplets of water (l-50nm in diameter) are surrounded by a monolayer of surfactant molecules (Fig. 9.15). The water pool inside the so-called reverse micelle represents a combination of properties of aqueous and non-aqueous environments. Enzymes entrapped inside reverse micelles depend in their catalytic activity on the size of the micelle, i.e. the water content of the system (at constant surfactant concentrations). 

Syntheses of uptake hydrogenases of cyanobacteria, as well as uptake hydrogenases of other bacteria, have been shown to be dependent on the availability of Ni in the growth medium. The enzymes are insensitive to O2 in whole-cell preparations, but become sensitive after cell extraction (Houchins and Burris 1981b). 

It is important to point out that D. vulgaris hydrogenase contains three multinuclear iron clusters and each cluster may exist in equilibrium between two different oxidation states in each sample. Consequently, the raw Mossbauer spectra are complex, consisting of overlapping spectra originating from different iron sites of these various clusters. For clarity, we present only the deconvoluted spectra of the H cluster. These spectra were prepared by removing the contributions of other iron species from the raw spectra. Details of the analysis are available (Pereira et al. 2001). 

Figure 4.2. Mossbauer spectrum of Hox+i hatched marks) prepared from the spectra of the aerobically purified D. vulgaris hydrogenase recorded at 4.2 K in a magnetic field of 0.05 T applied parallel to the y-rays. Solid line, a least-squares fit to the data, assuming three equal-intensity quadrupole doublets dotted-and-dashed line, doublet corresponding to the [2Fe]n cluster dashed line, superposition of two doublets (doublets 1 and 2) corresponding to the [4Fe-4S]n cluster.

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