Regeneration hydrogenases

Moreover, an electron transfer chain could be reconstituted in vitro that is able to oxidize aldehydes to carboxylic acids with concomitant reduction of protons and net production of dihydrogen (213, 243). The first enzyme in this chain is an aldehyde oxidoreductase (AOR), a homodimer (100 kDa) containing one Mo cofactor (MOD) and two [2Fe—2S] centers per subunit (199). The enzyme catalytic cycle can be regenerated by transferring electrons to flavodoxin, an FMN-con-taining protein of 16 kDa (and afterwards to a multiheme cytochrome and then to hydrogenase) ... 

The proposed mechanism of H2 evolution by a model of [FeFeJ-hydrogenases based upon DFT calculations [204-206] and a hybrid quanmm mechanical and molecular mechanical (QM/MM) investigation is summarized in Scheme 63 [207]. Complex I is converted into II by both protonation and reduction. Migration of the proton on the N atom to the Fe center in II produces the hydride complex III, and then protonation affords IV. In the next step, two pathways are conceivable. One is that the molecular hydrogen complex VI is synthesized by proton transfer and subsequent reduction (Path a). The other proposed by De Gioia, Ryde, and coworkers [207] is that the reduction of IV affords VI via the terminal hydride complex V (Path b). Dehydrogenation from VI regenerates I. 

Nonenzymatic regeneration of NAD(P)H requires the regioselective transfer of two electrons and a proton to NAD(P)+. Various rhodium(III) complexes are effective electrocatalysts capable of mimicking hydrogenase enzymes.48-54... 

In an early report to a process using three oxidoreductases, namely hydrogenase (ECl.12.2.1), lipoamide dehydrogenase (EC 1.6.4.3) and 20(3-hydroxysteroid dehydrogenase (ECl.1.1.53), a reverse micelle system was used to facilitate stereo- and site-specific reduction of apolar ketosteroids, assisted by the in situ NADH-regenerating enzyme system [61]. 

The enzyme hydrogenase (hydrogen dehydrogenase EC 1.12.1.2) is able to reduce electron acceptors by molecular hydrogen. When it is used in cofactor regenerating systems, consumed NADH can be regenerated directly by molecular hydrogen. 

Fig. 6 Use of ferredoxin-hydrogenase for NADPH regeneration here in a system for the stereoselective reduction of (2S)-hydroxy-l-phenyl-propanone to (lfi,2S)-phenyl-propane-l,2-diol catalyzed by ADH...

Fig. 27 Conversion of a-ketoglutarate to L-glutamate catalyzed by GluDH with coupled electrochemical regeneration of NADH using hydrogenase from Alcaligenes eutrophus...

The electroenzymatic reduction of NAD+ was successfully coupled with a synthesis reaction [122]. Hydrogenase from A. eutrophus was applied to regenerate NADH electrochemically during the transformation of a-ketoglutarate into L-glutamate catalyzed by an L-glutamate dehydrogenase (Fig. 27). 

Fig. 32 Reduction of cyclohexanone catalyzed by HLADH with simultaneous hydrogenase-driven regeneration of NADH in an organic-aqueous two-phase system...

Several organic solvents were investigated with regard to stability and activity of HLADH as well as their influence on the hydrogenase-driven reaction. Hydrophobic solvents such as heptane or toluene proved to be the most suitable solvents for the coupled enzyme-system. Furthermore, it became apparent that nonimmobilized cells, permeabilized with cetyl-trimethylammonium bromide, showed the best results for NADH regeneration. After optimization the conversion in heptane with 10% water yields 99% cyclohexanol by reduction of cyclohexanone. 

Figure 33. Regeneration of NADH by a photochemical system consisting of CdS-hydrogenase (from Alcaligenes eutrophus) and using formate as a sacrificial electron donor.

Figure 11. Hydrogenase-catalyzed NADH regeneration coupled to the reductive amination of a-ketoglutarate to L-glutmate catalyzed by L-glutamate dehydrogenase.

Nia-X intermediate, which undergoes two successive proton-coupled electron transfer steps to regenerate Nia-C (Lill and Siegbahn, 2009). The Ni—Fe hydrogenases also contain multiple Fe—S clusters which channel electrons to the catalytic site. 

Some oxidoreductases require nicotine adenine dinucleotide (NADH) as a cofactor.146 To use them in organic synthesis, as in the reduction of a ketone to an alcohol, it is necessary to have an efficient system to continuously regenerate them. A common way is to include in the same reaction formic acid and formate dehydrogenase, the byproduct being carbon dioxide.147 The regeneration of the cofactor can also be done electrochemically with or without the addition of a hydrogenase.148 The use of whole organisms eliminates this need. 

Figure 2 shows two different hypothetical catalytic cycles for [FeFe]-hydrogenases these two cycles differ primarily in whether the Hsred state is included as a catalytically relevant state [16, 17]. Despite this, there are a number of commonalities shared by both schemes. First, for proton reduction, both cycles start with a one-electron reduction of Hox to form Hred followed by protonation. It is unknown whether, during this step, the electron transfer or the proton transfer occurs first, or if they happen simultaneously. Second, both involve formation of a terminal hydride on the distal iron. Third, dihydrogen forms via combination of this hydride with a proton associated with the bulkhead nitrogen atom. Finally, in both cases, the H2 then dissociates to regenerate the Hqx state and complete the... 

Scheme 2.3 Regeneration of NADH cofactor in the lactate dehydrogenase (LDH)-catalyzed reduction of pyruvate, using Hj and pyrolytic graphite (PG) particles modified with a hydrogenase (Hyd-2) and a diaphorase (HoxFU) (see text for details).

---