Cofactors fixed

We next focus on the use of fixed-site cofactors and coenzymes. We note that much of this coenzyme chemistry is now linked to very local two-electron chemistry (H, CH3", CH3CO-, -NH2,0 transfer) in enzymes. Additionally, one-electron changes of coenzymes, quinones, flavins and metal ions especially in membranes are used very much in very fast intermediates of twice the one-electron switches over considerable electron transfer distances. At certain points, the chains of catalysis revert to a two-electron reaction (see Figure 5.2), and the whole complex linkage of diffusion and carriers is part of energy transduction (see also proton transfer and Williams in Further Reading). There is a variety of additional coenzymes which are fixed and which we believe came later in evolution, and there are the very important metal ion cofactors which are separately considered below. 

Nitrogenases from various nitrogen fixing organisms seem to contain the same Fe/Mo/S structural unit that occurs as an extractable cofactor (FeMoco) (2). Extracts of the Fe-Mo component protein from inactive mutant strains of different microorganisms that do not contain the Fe/Mo/S center are activated upon addition of the FeMoco. 

This has been isolated from a range of nitrogen-fixing organisms, and appears to be identical in all cases except when an inactive form is produced by mutants deficient in certain of the nif genes. It differs from the molybdenum cofactor found in a number of molybdoenzymes (Section 62.1.9). 

A large number of synthetic metal-sulfur cluster analogs have been prepared and some proposed as suitable models for FeMoco (see Section II). Some of the analogs exhibit spectroscopic properties very similar to those of the cofactors, but none shows nitrogen fixing ability. 

Multi-layer enzyme sensors offer the opportunity to separate the signals of different analytes. For this purpose a cofactor dependent enzyme converting the analyte A is fixed in front of a layer which contains enzymes converting both analytes B and A under the formation of an electrode-active product. Two procedures for separating the signals for both analytes have been established. 

The ultimate way to control diffusion and reactivity with redox partners is to restrict diffusion by anchoring a portion of the redox molecule and allowing essentially only one-dimensional diffusion. This is effectively the case for the FeS center of the bc complex, which has a mobile head group with a surface-exposed FeS center, but also a transmembrane anchor secured to the membrane portion of the bc complex. This severely restricts the range of motion ( 16 A) but perfectly controls the problem of guiding electron transfer. Diffusion over this distance should be on the submicrosecond time scale, much faster than the catalytic turnover of the complex. In a certain sense, this restricted diffusion has properties that lie between unrestricted diffusion and fixed redox cofactor chains a sort of chain with moving parts. 

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