NiFe catalytic cycle

The discovery of a new heterodinuclear active site in [NiFe] hydro-genases opens the way for the proposal of catalytic cycles based on the available spectroscopic data on the different active site redox states, namely EXAFS studies that reveal that the Ni-edge energy upon reduction of the enzyme supports an increase in the charge density of the nickel (191). 

We should also remember that not all of the states that we see when freezing the enzyme (Section 7.4) are necessarily part of the mechanism. The most stable enzyme molecule is a dead one, so we must be aware that some of the spectroscopic signals represent damaged molecules. In the [NiFe] hydrogenases, the NiA and NiB states probably are not involved in the catalytic cycle, because they react slowly, if at all, with H2. In the mechanism shown in Fig. 8.3, it is assumed that the relevant active states are NiSR, NiA and NiR. 

As ascribed, the EPR spectrum with g = 2.10 can be low-spin Fec(III). When the isolated enzyme is reductively titrated this signal disappears at a potential Emj -0.3 V [65]. This would seem to indicate that the putative Fec(III) form is not relevant, at least not to hydrogen-production activity. The cubane is a one-electron acceptor as it can shuttle between the 2+ and 1 + oxidation states. Therefore, if the active center were to take up a total of two electrons, then the oxidation state of the Fec would, as least formally, shuttle between II and I. Recently, a redox transition in Fe hydrogenase with an Em below the H2/H+ potential has been observed in direct electrochemistry [89]. This superreduced state has not been studied by spectroscopy. It might well correspond to the formal Fec(I) state. For NiFe hydrogenases Fec(I) has recently been proposed as a key intermediate in the catalytic cycle [90] (cf. Chapter 9). 

Fig. 8. Suggested catalytic cycle for [NiFe] hydrogenase with an uncharged His77.

Fig. 10. Energetics for the suggested catalytic cycle for [NiFe] hydrogenase. The numbers for the structures are those from Fig. 9. The full line corresponds to a charged His77, while the dashed line is for an uncharged His77.

The actual catalytic cycle of [NiFe] hydrogenase encompasses only three states Ni-SIa, Ni-C and Ni-R, which are interconverted by one-electron/one-proton equilibria (Figure 3.4.7A) [123, 124], In the catalytic process, the approaching H2 is attached to the Ni, and the bond is polarized followed by base-assisted heterolytic cleavage of the H2 molecule leading to a bridging hydride species. One of the candidates for acting as a base is a terminal cysteine at the Ni. Alternatively, a water molecule bound to the iron has been proposed [120]. Concomitant electron transfer to the proximal FeS cluster then leads to the Ni-C state, which has been shown to... 

Pandelia ME, Ogata H, Lubitz W. Intermediates in the catalytic cycle of [NiFe] hydrogenase functional spectroscopy of the active site. ChemPhysChem. 2010 11(6) 1127-40. 

Pardo A, de Lacey AL, Fernandez VM, Fan H. J, Fan Y, Hall MB. Density functional study of the catalytic cycle of nickel-iron [NiFe] hydrogenases and the involvement of high-spin nickel(II). J Biol Inorg Chem. 2006 ll(3) 286-306. 

The mechanism of the NiFe hydrogenase has been treated by caleula-tional methods, with some interesting conclusions (Pavlov et al., 1998). Scheme 1 of this reference proposes a catalytic cycle based on these results. It was proposed that Fe binds H2 and that a low spin Fe is essential for het-erolytic cleavage of the H6H bond. The next step is proposed to be hydride transfer to Fe and proton transfer to a ligated cysteine thiolate, whieh leads to decoordination of the cysteine and concurrent bridging of the N of CN... 

The nickel site has been proposed to be redox active and changes between Ni(III) and Ni(II), while the iron site remains as Fe(II) in all spectrally defined redox states of the enzyme. " The EXAFS/EPR studies indicate that the formal oxidation state of the Ni center is paramagnetic Ni(III) in Ni-A, Ni B and Ni-C states. Actually, the active form Ni-C (the paramagnetic Ni-C intermediate) of [NiFe] H2ase was proposed to exist as the [(Scys-H)Ni -H -Fe] intermediates after an active state Ni Sla (silent-active [(Scys-H)Ni (Scys)3]) is passed. Ni-C is beheved to be an intermediate in the catalytic cycle. Upon illumination. 

A summary of most of the known states of standard [NiFe] hydrogenases and their proposed relationships is depicted in Fig. 2. Four S = 1/2 paramagnetic states have been detected by EPR they are called Ni - A, Ni - B, Ni - C and Ni - L because the spin density is mainly localized on the Ni. Five diamagnetic states, called Ni-SU, Ni- S , Ni-SI, SI-CO and Ni-R, have been characterized by FTIR, thanks to the vibration bands of the triple bonds in the CO and CN ligands found in the 1900-2100 cm range. The active Ni - C and Ni-R states are directly involved in the catalytic cycle whereas the unready Ni - A and Ni - SU states are inactive and require a long reductive activation. The ready Ni - B and Ni - SI states are also inactive but can be immediately activated by H2 in the absence of O2. An additional active... 

Figure 3 shows a proposed catalytic mechanism for [NiFe]-hydrogenases. For hydrogen oxidation, the catalytic cycle starts at the Ni-SU state, a Ni(II) state with no bridging ligand. Hydrogen binds the active site and is heteroly tic ally cleaved to produce the Ni-R state, a Ni(II) species with a bridging hydride and likely a... 

F. 3 The catalytic cycle of NiFe]-hydrogenases. Reprinted with permission from Lubitz... 

Catalytic Cycle of the [NiFe] Hydrogenases. Figure 3A shows a simplified diagram of the sites of electron and proton transfer in [NiFe]-hydrogenase. Figure 4 shows the reaction with H2. The consumption of H2 by the enzyme is the reverse of this process. The steps are indicated by circled numbers. 

Figure 2. Overview of the redox states of [NiFe] hydrogenases, from the most oxidized (top) to the most reduced (bottom) form. Indicated are the IR frequencies (IR) of the CO ligand and the two CN ligands to Fe for D. vulgaris Miyazaki F hydrogenase and the midpoint potentials ( ) far the redox transitions at pH = 6 (Ni-A/NiSU) and at pH = 7.4 (all others). The paramagnetic states are given in bold face, the EPR-silent states in italics. The two states NiSIr and NiSIa are in an acid-base equilibrium. The states involved directly in the catalytic cycle are highlighted by a shaded box. The paramagnetic Ni-CO state (not shown) is probably derived from the Ni-L state.

The EPR studies on the paramagnetic states [98]— together with other investigations using, for example, FTIR and XAS studies that can be applied to the EPR-silent states—have delivered important insight into the catalytic cycle of [NiFe] hydrogenase as well as in activation/deactivation and inhibition of this important enzyme. However, the picture is still far from being complete. 

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