Enzyme Electron Transfer

The possibility that there might be long-range electron transfer between redox-active centers in enzymes was first suspected by biochemists working on the mechanism of action of metalloenzymes such as xanthine oxidase which contain more than one metal-based redox center. In these enzymes electron transfer frequently proceeds rapidly but early spectroscopic measurements, notably those by electron paramagnetic resonance, failed to provide any indication that these centers were close to one another. 

Although ferrocenes have shown themselves to be excellent mediators of enzyme electron transfer in solution, they need to be incorporated into a fabrication process to produce sensors that are suitable for clinical use. The demands of implanted sensors are quite different from those that typically use a fingerstick for ex vivo measurement." The former is described later in this review this section covers screen printed sensors. The process of screen printing lends itself well to the manufacture of disposable, single-use sensorsand has been widely employed for this in both the laboratory and in large-scale manufacture. 

Enzymes with flavin coenzymes in human metabolism (examples of 100 enzymes). Electron-transfer flavo-protein (EC 1.3.99.2-3) EAD NADPH-cyto-chrome-P-450-reductase (EC 1.6.2.4) EMN and FAD succinate dehydrogenase (EC 1.3.99.1) EAD D-amino acid oxidase (EC 1.4.3.3) EAD L-amino acid oxidase (EC 1.4.3.2) FMN. 

The use of electrochemical methods to smdy protein and enzyme electron transfer reaction kinetics, thermodynamics, and mechanisms directly with electrodes is becoming a mature field. Twenty years ago such studies were rarely conducted outside of laboratories with substantial experience in electrochemistry. Now scientists in diverse fields have taken up cyclic voltammetry, square wave voltammetry, and other electrochemical methods to study biological systems. Clearly much has been learned about how to conduct reliable electrochemical experiments on complex biological samples using direct electron transfer at electrodes. Progress in this field was slow, and some background is provided to put the current state of this field in context. 

It appears that the biological nitrogen fixation is mainly dependent on two proteins containing transition metals in cells i.e. nitrogenase system consisting of Mo-Fe protein and Fe protein, and electron-donator (biological reductants and corresponding enzymes), electron transfer (e.g. Fe-O protein, flavoenzyme or methyl violet) as well as ATP-Mg2+ etc. ... 

The study of fundamental electron transfer processes at nanoelectrodes has also been extended to the field of bioelectrochemistry, notably in the elucidation of enzyme electron transfer kinetics and mechanism via protein film voltammetry. This typically involves immobilizing a film of redox active enzymes onto an electrode such that electronic contact is achieved between the enzyme active site and the underlying surface, enabling voltammetry to... 

Copper Cu 1.5-3 mg Oxidative enzymes, electron-transferring proteins... 

Iron Sulfur Compounds. Many molecular compounds (18—20) are known in which iron is tetrahedraHy coordinated by a combination of thiolate and sulfide donors. Of the 10 or more stmcturaHy characterized classes of Fe—S compounds, the four shown in Figure 1 are known to occur in proteins. The mononuclear iron site REPLACE occurs in the one-iron bacterial electron-transfer protein mbredoxin. The [2Fe—2S] (10) and [4Fe—4S] (12) cubane stmctures are found in the 2-, 4-, and 8-iron ferredoxins, which are also electron-transfer proteins. The [3Fe—4S] voided cubane stmcture (11) has been found in some ferredoxins and in the inactive form of aconitase, the enzyme which catalyzes the stereospecific hydration—rehydration of citrate to isocitrate in the Krebs cycle. In addition, enzymes are known that contain either other types of iron sulfur clusters or iron sulfur clusters that include other metals. Examples include nitrogenase, which reduces N2 to NH at a MoFe Sg homocitrate cluster carbon monoxide dehydrogenase, which assembles acetyl-coenzyme A (acetyl-CoA) at a FeNiS site and hydrogenases, which catalyze the reversible reduction of protons to hydrogen gas. 

A compound which is a good choice for an artificial electron relay is one which can reach the reduced FADH2 active site, undergo fast electron transfer, and then transport the electrons to the electrodes as rapidly as possible. Electron-transport rate studies have been done for an enzyme electrode for glucose (G) using interdigitated array electrodes (41). The following mechanism for redox reactions in osmium polymer—GOD biosensor films has... 

The next generation of amperomethc enzyme electrodes may weU be based on immobilization techniques that are compatible with microelectronic mass-production processes and are easy to miniaturize (42). Integration of enzymes and mediators simultaneously should improve the electron-transfer pathway from the active site of the enzyme to the electrode. 

Most of the Moco enzymes catalyze oxygen atom addition or removal from their substrates. Molybdenum usually alternates between oxidation states VI and IV. The Mo(V) state forms as an intermediate as the active site is reconstituted by coupled proton—electron transfer processes (62). The working of the Moco enzymes depends on the 0x0 chemistry of Mo (VI), Mo(V), and Mo (TV). 

The most conspicuous use of iron in biological systems is in our blood, where the erythrocytes are filled with the oxygen-binding protein hemoglobin. The red color of blood is due to the iron atom bound to the heme group in hemoglobin. Similar heme-bound iron atoms are present in a number of proteins involved in electron-transfer reactions, notably cytochromes. A chemically more sophisticated use of iron is found in an enzyme, ribo nucleotide reductase, that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an important step in the synthesis of the building blocks of DNA. 

Further improvements can be achieved by replacing the oxygen with a non-physiological (synthetic) electron acceptor, which is able to shuttle electrons from the flavin redox center of the enzyme to the surface of the working electrode. Glucose oxidase (and other oxidoreductase enzymes) do not directly transfer electrons to conventional electrodes because their redox center is surroimded by a thick protein layer. This insulating shell introduces a spatial separation of the electron donor-acceptor pair, and hence an intrinsic barrier to direct electron transfer, in accordance with the distance dependence of the electron transfer rate (11) ... 

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