Electrochemical studies of the hexaheme nitrite reductase from Desulfovibrio desulfuricans ATCC 27774

Abstract

The electron-transfer kinetics between three different mediators and the hexahemic enzyme nitrite reductase isolated from Desulfovibrio desulfuricans (ATCC 27774) were investigated by cyclic voltammetry and by chronoamperometry. The mediators, methyl viologen, Desulfovibrio vulgaris (Hildenborough) cytochrome c3 and D. desulfiricans (ATCC 27774) cytochrome c3 differ in structure, redox potential and charge. The reduced form of each mediator exchanged electrons with nitrite reductase. Second-order rate constants, k, were calculated on the basis of the theory for a simple catalytic mechanism and the results, obtained by cyclic voltammetry, were compared with those obtained by chronoamperometry. Values for k are in the range lo6-lo8 M-' s-' and increase in the direction D. desulfiricans cytochrome c3 -+ D. vulgaris cytochrome c3 + methyl viologen.An explanation is advanced on the basis of electrostatic interactions and relative orientation between the partners involved.Chronoamperometry (computer controlled) offers advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate and better reproducibility).Direct, unmediated electrochemical responses of the hexaheme nitrite reductase were also reported.The study of electron-transfer processes in biological systems by electrochemical methods has recently received increasing attention. For a number of a small-sized redox proteins (e.g. cytochrome c, cytochrome c3 and ferredoxins) a direct electron transfer at metal oxide electrodes [l] or at carbon electrodes [2,3] has been achieved. The electrode kinetics of these systems can be investigated by such standard techniques as cyclic voltammetry, rotating-disk electrode or impedance measurements. Still, for larger-sized redox enzymes, no direct electron transfer at electrodes has yet been described. Reports of direct electron transfer between redox enzymes and electrodes remain scarce. Some examples are studies of hydrogenase at the dropping mercury electrode in the presence of polylysine [4], covalently modified glucose oxidase at metal electrodes [5] and cytochrome c peroxidase at tin oxide electrodes [6].Coupling an enzyme to an electrochemical reaction by use of redox mediators offers an alternative approach. Voltammetric and chronoamperometric methods can be used to measure the homogeneous electron transfer rates between redox enzymes and artificial mediators or between redox en-

A needle in a haystack: The active site of the membrane‐bound complex cytochrome c nitrite reductase

“…In the presence of substrate, the catalytic process results in a steady-state electron flow whose detection allows instantaneous measurement of turnover rates. Catalytic electrochemical signals have been reported independently for the ccNiRs from D. desulfuricans [13] and E. coli [14,15]. Here we shall report on the former enzyme.…”

Protein Film Voltammetry Reveals Distinctive Fingerprints of Nitrite and Hydroxylamine Reduction by a Cytochrome c Nitrite Reductase

“…The differences between the absolute responses recorded in each substrate will reflect differences in the rate constants and reduction potentials specific to the catalytic cycle describing reduction of each substrate. Interestingly, our inspection, of the solution voltammetry of D. desulfuricans cytochrome c nitrite reductase under conditions supporting a maximum nitrite reduction rate and measured with a view to development of a nitrite biosensor, shows a catalytic waveform of similar shape to that observed during PFV of the E. coli enzyme (19,21). This suggests that the description of enzyme-limited substrate reduction uncovered by PFV will be common to the cytochrome c nitrite reductases, in agreement with the conservation of active site structure and heme packing motifs recently revealed by structural studies of this important family of enzymes.…”

Structure of cytochrome c nitrite reductase

“…deleyianum cytochrome c nitrite reductase has a high speci®c activity, with up to 1,050 mmol NO -2 per min per mg (pH 7); this has led to the development of an amperometric biosensor 15,16 . The enzyme from S. deleyianum also reduced sulphite to hydrogen sulphide, again a six-electron transfer process 17 .…”