Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA 2 NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.

From the bench-mark work on microfluidics from the Whitesides’s group in 2007, paper technology has experienced significant growth, particularly regarding applications in biomedical research and clinical diagnostics. Besides the structural properties supporting microfluidics, other advantageous features of paper materials, including their versatility, disposability and low cost, show off the great potential for the development of advanced and eco-friendly analytical tools. Consequently, paper was quickly employed in the field of electrochemical sensors, being an ideal material for producing custom, tailored and miniaturized devices. Stencil-, inkjet-, or screen-printing are the preferential techniques for electrode manufacturing. Not surprisingly, we witnessed a rapid increase in the number of publications on paper based screen-printed sensors at the turn of the past decade. Among the sensing strategies, various biosensors, coupling electrochemical detectors with biomolecules, have been proposed. This work provides a critical review and a discussion on the future progress of paper technology in the context of miniaturized printed electrochemical biosensors.

ARTICLE This journal isBsDyP from Bacillus subtilis belongs to the new dye-decolourising peroxidase (DyP) family. Here we use transient kinetics to provide details on the catalytic cycle of BsDyP. The reaction of BsDyP with H 2 O 2 exhibits saturation behaviour consistent with a two-step mechanism involving the formation of an E-H 2 O 2 intermediate (K 1 = (12 ± 1) × 10 -6 M) followed by formation of Compound I (k 1 = 22 ± 1 s -1 ). We demonstrate that the k 1obs is pH-dependent and controlled by an ionisable group with a pK a of 4.3 suggesting the involvement of distal Asp. The reaction of Compound I with guaiacol obeys second order kinetics (k 3 ' = (0.21 ± 0.01) × 10 6 M -1 s -1 ) while the reaction of Compound II with guaiacol shows saturation kinetics (K 4 = 22 ± 5) × 10 -6 M and k 4 = 0.13 ± 0.01 s -1 ) and is the rate-limiting step in the BsDyP catalytic cycle. We furthermore use transient and steady-state kinetics, spectroscopic and electrochemical approaches to investigate the role of distal Asp240, Arg339 and Asn244 and proximal Asp383 residues in BsDyP. All mutations of distal residues affect particularly the K 1 (and K m ) for H 2 O 2 leading to catalytic efficiencies (k cat /K m ) of only one to two orders of magnitude lower than in the wild type. Notably, a significant improvement in the catalytic efficiency for reducing substrates is observed in variants. We conclude that the Asp and Arg residues are important for the proper binding of H 2 O 2 to the haem but none is individually indispensable for promoting H 2 O 2 (de)protonation and O-O bond cleavage. The obtained kinetic data suggest an important role of the distal Asn in modulating the acidbase catalysis of BsDyP. Our findings contribute to the establishment of structural determinants of DyPs that underlie their mechanistic properties; this has implications for their potential in biotechnological applications and sheds more light on subfamily-dependent features of these enzymes.