M. Gabriela Almeida

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

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 NrfA2NrfH 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.

An efficient non-mediated amperometric biosensor for nitrite determination

In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.

A novel nitrite biosensor based on conductometric electrode modified with cytochrome c nitrite reductase composite membrane

A conductometric biosensor for nitrite detection was developed using cytochrome c nitrite reductase (ccNiR) extracted from Desulfovibrio desulfuricans ATCC 27774 cells immobilized on a planar interdigitated electrode by cross-linking with saturated glutaraldehyde (GA) vapour in the presence of bovine serum albumin, methyl viologen (MV), Nafion, and glycerol. The configuration parameters for this biosensor, including the enzyme concentration, ccNiR/BSA ratio, MV concentration, and Nafion concentration, were optimized. Various experimental parameters, such as sodium dithionite added, working buffer solution, and temperature, were investigated with regard to their effect on the conductance response of the biosensor to nitrite. Under the optimum conditions at room temperature (about 25 degrees C), the conductometric biosensor showed a fast response to nitrite (about 10s) with a linear range of 0.2-120 microM, a sensitivity of 0.194 microS/microM [NO(2)(-)], and a detection limit of 0.05 microM. The biosensor also showed satisfactory reproducibility (relative standard deviation of 6%, n=5). The apparent Michaelis-Menten constant (K(M,app)) was 338 microM. When stored in potassium phosphate buffer (100mM, pH 7.6) at 4 degrees C, the biosensor showed good stability over 1 month. No obvious interference from other ionic species familiar in natural waters was detected. The application experiments show that the biosensor is suitable for use in real water samples.

Small electron-transfer proteins as mediators in enzymatic electrochemical biosensors

Electrochemical mediators transfer redox equivalents between the active sites of enzymes and electrodes and, in this way, initiate bioelectrocatalytic redox processes. This has been very useful in the development of the so-called second-generation biosensors, in which they transduce a catalyzed reaction into an electrical signal. Among other pre-requisites, redox mediators must be readily oxidized and/or reduced at the electrode surface and readily interact with the biorecognition component. Small chemical compounds (e.g. ferrocene derivatives, ruthenium, or osmium complexes and viologens) are frequently used for this purpose but, lately, small redox proteins (e.g. horse heart cytochrome c) have also been used as redox partners in biosensing applications. In general, docking between two complementary proteins introduces a second level of selectivity to the biosensor and enlarges the list of compounds analyzed. Moreover, electrochemical interferences are frequently minimized owing to the small overpotentials achieved. This paper provides an overview of enzyme biosensors that are mediated by electron-transfer proteins. The paper begins with a brief discussion of mediated electrochemistry in biosensing systems and proceeds with a detailed description of relevant work on the cooperative use of redox enzymes and biological electron donors and/or acceptors.

SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes

Cytochrome cd1 nitrite reductases (cd 1NiRs) catalyze the one-electron reduction of nitrite to nitric oxide. Due to their catalytic reaction, cd 1NiRs are regarded as promising components for biosensing, bioremediation and biotechnological applications. Motivated by earlier findings that catalytic activity of cd 1NiR from Marinobacter hydrocarbonoclasticus (Mhcd 1) depends on the presence of its physiological redox partner, cytochrome c 552 (cyt c 552), we show here a detailed surface enhanced resonance Raman characterization of Mhcd 1 and cyt c 552 attached to biocompatible electrodes in conditions which allow direct electron transfer between the conducting support and immobilized proteins. Mhcd 1 and cyt c552 are co-immobilized on silver electrodes coated with self-assembled monolayers (SAMs) and the electrocatalytic activity of Ag // SAM // Mhcd 1 // cyt c 552 and Ag // SAM // cyt c 552 // Mhcd 1 constructs is tested in the presence of nitrite. Simultaneous evaluation of structural and thermodynamic properties of the immobilized proteins reveals that cyt c 552 retains its native properties, while the redox potential of apparently intact Mhcd 1 undergoes a ~150 mV negative shift upon adsorption. Neither of the immobilization strategies results in an active Mhcd 1, reinforcing the idea that subtle and very specific interactions between Mhcd 1 and cyt c 552 govern efficient intermolecular electron transfer and catalytic activity of Mhcd 1.

Electron transfer and docking between cytochrome cd1 nitrite reductase and different redox partners - A comparative study.

Cytochrome cd1 nitrite reductases (cd1NiRs) catalyze the reduction of nitrite to nitric oxide in denitrifying bacteria, such as Marinobacter hydrocarbonoclasticus. Previous work demonstrated that the enzymatic activity depends on a structural pre-activation triggered by the entry of electrons through the electron transfer (ET) domain, which houses a heme c center. The catalytic activity of M. hydrocarbonoclasticus cd1NiR (Mhcd1NiR) was tested by mediated electrochemistry, using small ET proteins and chemical redox mediators. The rate of enzymatic reaction depends on the nature of the redox partner, with cytochrome (cyt) c552 providing the highest value. In situations where cyt c552 is replaced by either a biological (cyt c from horse heart) or a chemical mediator the catalytic response was only observed at very low scan rates, suggesting that the intermolecular ET rate is much slower. Molecular docking simulations with the 3D model structure of Mhcd1NiR and cyt c552 or cyt c showed that hydrophobic interactions favor the formation of complexes where the heme c domain of the enzyme is the principal docking site. However, only in the case of cyt c552 the preferential areas of contact and Fe-Fe distances between heme c groups of the redox partners allow establishing competent ET pathways. The coupling of the enzyme with chemical redox mediators was also found not to be energetically favorable. These results indicate that although low activity functional complexes can be formed between Mhcd1NiR and different types of redox mediators, efficient ET is only observed with the putative physiological electron donor cyt c552.

Understanding the response of Desulfovibrio desulfuricans ATCC 27774 to the electron acceptors nitrate and sulfate - biosynthetic costs modulate substrate selection

Sulfate-reducing bacteria (SRB) are a diverse group of anaerobic microorganisms that obtain their energy from dissimilatory sulfate reduction. Some SRB species have high respiratory versatility due to the possible use of alternative electron acceptors. A good example is Desulfovibrio desulfuricans ATCC 27774, which grows in the presence of nitrate (end product: ammonium) with higher rates and yields to those observed in sulfate containing medium (end product: sulfide). In this work, the mechanisms supporting the respiratory versatility of D. desulfuricans were unraveled through the analysis of the proteome of the bacterium under different experimental conditions. The most remarkable difference in the two-dimensional gel electrophoresis maps is the high number of spots exclusively represented in the nitrate medium. Most of the proteins with increase abundance are involved in the energy metabolism and the biosynthesis of amino acids (or proteins), especially those participating in ammonium assimilation processes. qPCR analysis performed during different stages of the bacteriums growth showed that the genes involved in nitrate and nitrite reduction (napA and nrfA, respectively) have different expressions profiles: while napA did not vary significantly, nrfA was highly expressed at a 6h time point. Nitrite levels measured along the growth curve revealed a peak at 3h. Thus, the initial consumption of nitrate and concomitant production of nitrite must induce nrfA expression. The activation of alternative mechanisms for energy production, aside several N-assimilation metabolisms and detoxification processes, solves potential survival problems in adapting to different environments and contributes to higher bacterial growth rates.

Assessment of human paraoxonase activity by electrochemistry: a simple and novel approach

Human serum paraoxonase 1 (EC 3.1.8.1, PON1), a calcium dependent enzyme, is an endogenous free-radical scavenging system with arylesterase, lactonase and paraoxonase activities. The determination of PON1 activity has been gaining an increasing role in clinical diagnosis due to its possible relationship with atherosclerosis and derived diseases. The paraoxonase activity protects against xenobiotic toxicity and was the first to be discovered. It has been the most used activity to assess PON1 status, through the spectrophotometric measurement of the hydrolysis of organophosphate compounds, such as paraoxon. However, these methods are prone to interferences and require specialized equipment. Herein, a simple alternative electrochemical assay for the assessment of PON1 activity is proposed for the first time. The catalytic hydrolysis of paraoxon was monitored by square-wave voltammetry, and the kinetic parameters of Escherichia coli expressed PON1 variant G3C9 (Vappmax 530 ± 40 nM s−1, KappM 1.7 ± 0.4 mM) and native PON1 (Vappmax 390 ± 40 nM s−1, KappM 1.3 ± 0.3 mM), present in human plasma, were determined.

Nitrite Biosensing Using Cytochrome C Nitrite Reductase: Towards a Disposable Strip Electrode

This paper presents the results of a primary study that aims to produce miniaturized biosensing devices for nitrite analysis in clinical samples. Following our previous works regarding the development of amperometric nitrite biosensors using the nitrite reducing enzyme (ccNiR) from Desulfovibrio desulfuricans ATCC 27774, here we aimed at reducing the size of the experimental set-up according to the specific needs of biomedical applications. For this, thick-film strip electrodes made of carbon conductive inks deposited on plastic supports were modified with the ccNiR enzyme, previously mixed with the conductive graphite ink. Firstly, though, the electrode preparation was optimized (enzyme amount, organic solvent and curing temperature). Then, the biocompatibility of ccNiR with these harsh treatments and the analytical performance of the modified electrodes were evaluated by cyclic voltammetry. Finally, the carbon paste screen-printed electrodes were coated with the ccNiR/carbon ink composite, displaying a good sensitivity (5.3x10− 7 A.uM− 1.cm− 2) within the linear range of 0.001 - 1.5 mM.