Titration Transition state intermediates

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). 

Figure 4.13 pH-Titration of protein Hsp47 monitored by intrinsic tryptophan fluorescence spectroscopy (excitation 295 nm). Data is also indicative of a two stage transition between an Alkali stable state and an Acid stable state via a transient intermediate state (see Fig. 4.8). Fluorescence spectroscopy also reveals that Hsp47 will undergo reversible pFI-driven trans-conformational changes (see Chapter 7) (Reproduced from Momma et al., 2008). /em(iC) is fluorescence emission intensity.

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