Electrochemistry of Metalloproteins

In the early investigations of the direct electrochemistry of metalloproteins, polarography was principally employed. The electrode reaction of cytochrome c at mercury electrodes has been extensively stud- 

The electrochemistry of metalloproteins has developed markedly (4-6) over the past 15 years. It has mainly been concerned with the electrochemistry at solid electrodes gold, upon which are adsorbed redox-inactive promoters, i.e., molecules that bind both to the electrode and the protein and edge-plane graphite, with or without redox-inert metal ions in solution. 

Mitochondrial cytochrome c is perhaps the most widely studied of all metalloproteins with respect to its electrochemical properties. It is located in the inner-membrane space of mitochondria and transfers electrons between membrane-bound complex III and complex IV. The active site is an iron porphyrin with a redox potential (7) of -1-260 mV vs. NHE. The crystal structures of cytochrome c from tuna have been determined (8, 9) in both oxidation states at atomic resolution. It is found that the heme group is covalently linked to the protein via two thioether bridges, and part of its edge is exposed at the protein surface. Cytochrome c is a very basic protein, with an overall charge of -1-7/-l-8 at neutral pH. Furthermore, many of the excess basic lysine residues are clustered around the mouth of the heme crevice, giving rise to a pronounced charge asymmetry. 

One of the first reports on the quasireversible electrochemistry of redox proteins appeared in 1977 when Eddowes and Hill demonstrated (10) cyclic voltammetry of horse heart cytochrome c at a gold electrode in the presence of 4,4 -bipyridyl (Bipy) in solution. In the voltammo-grams (Fig. 1), the peak-to-peak separations were close to 60 mV and the faradaic currents varied linearly with (scan rate), indicating a quasireversible one-electron transfer process controlled by linear diffusion of redox species to the electrode surface. The midpoint potential 

A more detailed kinetic investigation of the Au/Bipy/cytochrome c system was carried out using the rotating ring-disk technique (12). It was found that rate constants for adsorption and desorption of the protein were 3 x cm sec and 50 sec , respectively. The limiting first-order rate constant within the protein-electrode complex was determined as 50 sec , a reasonable value as compared to that of long-range electron transfer between or within proteins. 

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As such, they are much more resistant to acid- or base-induced dissociation or hydrolysis than are coordination complexes [e.g., Fen(TPP) is stable in the presence of 1 M H30+ or 1 M HO", whereas Feu(NH3)4+ is not]. An especially useful review of the electrochemistry of metalloporphyrins in nonaqueous media is available,3 which is complemented by a review of the electrochemistry of metalloproteins.4... 

Eddowes, M.J. and Hill, H.A.O. (1977) Novel method for the investigation of the electrochemistry of metalloproteins cytochrome c. Journal of the Chemical Society. Chemical Communications,... 

In his excellent review, Armstrong [11] authoratively discussed the subject and emphasized the useful information that can be obtained from direct electrochemistry of metalloproteins. 

Gunner MR, Alexov E, Torres E et al (1997) The importance of the protein in controlling the electrochemistry of heme metalloproteins methods of calculation and analysis. J Biol Inorg... 

Finally, we must somehow resolve the vexing problem of the chemical speciation of trace elements in seawater what is the chemical nature of the various metal chelators whose existence has been demonstrated by electrochemistry Is the chemistry of several metals in surface seawater really controlled by metallophores released by prokaryotes Or are dissolved metals chiefly present as parts of metalloproteins in the process of remineralization How do metal chelators affect the residence times of metals (in particular, scavenged elements such as iron and cobalt) and in turn how do those chelators influence the global carbon cycle via changes in marine primary productivity ... 

The opportunity of obtaining direct electrochemistry of cytochrome c and other metalloproteins at various electrode materials such as modified gold and pyrolytic graphite has led to numerous reports of heterogeneous electron transfer rates and mechanisms between the protein and the electrode. In all the reports, Nicholson s method (37) was employed to calculate rate constants, which were typically within the range of 10" -10 cm sec with scan rates varying between 1 and 500 mV sec This method is based on a macroscopic model of the electrode surface that assumes that mass transport of redox-active species to and from the electrode occurs via linear diffusion to a planar disk electrode and that the entire surface is uniformly electroactive, i.e., the heterogeneous electron transfer reaction can take place at any area. 

Interfacial Electrochemistry of Redox Metalloproteins and Metalloenzymes Towards the Single-Molecule Level... 

Protein function at solid-liquid interfaces holds a structural and a dynamic perspective [31]. The structural perspective addresses macroscopic adsorption, molecular interactions between the protein and the surface, collective interactions between the individual adsorbed protein molecules, and changes in the conformational and hydration states of the protein molecules induced by these physical interactions. Interactions caused by protein adsorption are mostly non-covalent but strong enough to cause drastic functional transformations. All these features are, moreover, affected by the double layer and the electrode potential at electrochemical interfaces. Factors that determine protein adsorption patterns have been discussed in detail recently, both in the broad context of solute proteins at solid surfaces [31], and in specific contexts of interfacial metalloprotein electrochemistry [34]. Some important elements that can also be modelled in suitable detail would be ... 

Our short overview illustrates some prospects for investigation of metallop-rotein dynamics at metal-solution interfaces. Cyclic voltammetry of small single metalloproteins is straightaway feasible. Reversible electrochemistry can be achieved but molecular detail such as adsorption patterns and precise promoter function remain elusive. 

Investigations of multicentre electrochemical metalloprotein function including metalloenzyme function have also been brought to a level, where both direct and catalytic modes, and elements of molecular mechanisms can be addressed. The latter are, however, entangled by features such as composite electrochemistry, extremely complicated molecular interaction patterns when more than two metallic redox centres are involved, fragile surface enzyme preparations, and lack of structural surface characterization of the adsorbed metalloenzymes. In this respect, two-centre metalloproteins constitute interesting promising intermediates where the coop-erativity between the metallic redox centres can be accurately addressed with molecular resolution within reach. 

One line in bioelectrochemistry is rooted in electrochemical techniques, spectroscopy, and other physical chemical techniques. Linear and cyclic voltammetry are central.Other electrochemical techniques include impedance and electroreflectance spectroscopy," ultramicro-electrodes, and chronoamperometry. To this come spectroscopic techniques such as infiared, surface enhanced Raman and resonance Raman,second harmonic generation, surface Plasmon, and X-ray photoelectron spectroscopy. A second line has been to combine state-of-the-art physical electrochemistry with corresponding state-of-the-art microbiology and chemical S5mthesis. The former relates to the use of a wide range of designed mutant proteins, " the latter to chemical synthesis or de novo designed synthetic redox metalloproteins. " " ... 

Even accepting that an isolated metalloprotein is not different from what it was in its natural environment, the feasibility of direct electrochemistry is subject to three main kinds of limitations ... 

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