Electrochemical titrations and reaction time courses monitored in situ by magnetic circular dichroism spectroscopy

Magnetic circular dichroism (MCD) spectra, at ultraviolet–visible or near-infrared wavelengths (185–2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.     

Molecular interactions between multihaem cytochromes: probing the protein-protein interactions between pentahaem cytochromes of a nitrite reductase complex

The cytochrome c nitrite reductase NrfA is a 53?kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2- to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21?kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein-protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer-dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2- or HSO3-. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50?nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.     

Physiological function and catalytic versatility of bacterial multihaem cytochromes c involved in nitrogen and sulfur cycling

Bacterial MCCs (multihaem cytochromes c) represent widespread respiratory electron-transfer proteins. In addition, some of them convert substrates such as nitrite, hydroxylamine, nitric oxide, hydrazine, sulfite, thiosulfate or hydrogen peroxide. In many cases, only a single function is assigned to a specific MCC in database entries despite the fact that an MCC may accept various substrates, thus making it a multifunctional catalyst that can play diverse physiological roles in bacterial respiration, detoxification and stress defence mechanisms. The present article briefly reviews the structure, function and biogenesis of selected MCCs that catalyse key reactions in the biogeochemical nitrogen and sulfur cycles.     

Structural Basis of Biological NO Generation by Octaheme Oxidoreductases

Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N₂H₄). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.     

Spectopotentiometric properties and salt-dependent thermotolerance of a [2Fe-2S] ferredoxin-involved nitrate assimilation in Haloferax mediterranei

Haloferax mediterranei is a halophilic archaeon that can grow using nitrate as the sole nitrogen source. A ferredoxin that serves as the physiological electron donor to the nitrate and nitrite reductases in this assimilatory process has been characterized. The ferredoxin was found to contain approximately two atoms of iron and two atoms of sulphur, indicative of the binding of a [2Fe-2S] cluster. The electron paramagnetic resonance spectrum of the reduced form of the protein displayed a rhombic signal, with g(x)=1.91, g(y)=1.98, g(z)=2.07, that shows considerable similarity to plant and algal [2Fe-2S] ferredoxins. UV-visible spectropotentiometric analysis determined a midpoint redox potential for the [2Fe-2S](2+/1+) transition of around -285 mV vs. SHE that was independent of salt concentration. UV-visible spectroscopy was also used to establish that the [2Fe-2S] cluster integrity of this protein was maintained over the pH range 5-11. Significantly, the Haloferax mediterranei ferredoxin was shown to be a highly thermostable protein. It was stable up to 60 degrees C in a low-salt (0.2 M) medium and this increased to 80 degrees C in a high-salt (4 M) medium. This thermostability at high salt concentration is an essential physiological characteristic because haloarchaea are mainly found in environments where high temperatures and concentrated salt water occur.     

Contrasting catalytic profiles of multiheme nitrite reductases containing CxxCK heme-binding motifs

The multiheme cytochromes from Thioalkalivibrio nitratireducens (TvNiR) and Escherichia coli (EcNrfA) reduce nitrite to ammonium. Both enzymes contain His/His-ligated hemes to deliver electrons to their active sites, where a Lys-ligated heme has a distal pocket containing a catalytic triad of His, Tyr, and Arg residues. Protein-film electrochemistry reveals significant differences in the catalytic properties of these enzymes. TvNiR, but not EcNrfA, requires reductive activation. Spectroelectrochemistry implicates reduction of His/His-ligated heme(s) as being key to this process, which restricts the rate of hydroxide binding to the ferric form of the active-site heme. The K _M describing nitrite reduction by EcNrfA varies with pH in a sigmoidal manner that is consistent with its modulation by (de)protonation of a residue with pK _a ≈ 7.6. This residue is proposed to be the catalytic His in the distal pocket. By contrast, the K _M for nitrite reduction by TvNiR decreases approximately linearly with increase of pH such that different features of the mechanism define this parameter for TvNiR. In other regards the catalytic properties of TvNiR and EcNrfA are similar, namely, the pH dependence of V _max and the nitrite dependence of the catalytic current–potential profiles resolved by cyclic voltammetry, such that the determinants of these properties appear to be conserved.     

Magnetic circular dichroism of hemoproteins with in situ control of electrochemical potential: "MOTTLE"

Hemoproteins have been recognized for nearly a century and are ubiquitous components of cellular organisms. Despite our familiarity with these proteins, defining the functional role of a given heme can still present considerable challenges. In this situation, magnetic circular dichroism (MCD) is a technique of choice because it has the capacity to define heme oxidation, spin, and ligation states in solution and at ambient temperature. Unfortunately, the resolving power of MCD rarely has been brought to bare on the intermediate redox states accessible to multiheme proteins. This is due in large part to the time-consuming procedure of magnetic field cycling required each time a sample is introduced into the magnet and the risk that control over, and knowledge of, the potential will be lost between sample preparation and spectral acquisition. Here we present a solution to this problem in the form of MCD-compatible optically transparent thin-layer electrochemistry (MOTTLE). MOTTLE defines redox behavior for cytochrome c in good agreement with the literature. In addition, MOTTLE reproduces the redox-driven transformation of heme ligand sets reported for cytochrome bd. Thus, MOTTLE provides a robust analytical tool for the dissection of heme properties with resolution across the electrochemical potential domain.     

Resolving complexity in the interactions of redox enzymes and their inhibitors: contrasting mechanisms for the inhibition of a cytochrome c nitrite reductase revealed by protein film voltammetry

Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium. The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV). Azide inhibition is rapid and reversible. Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (approximately 40 mM) with a lower affinity than to the enzyme alone (approximately 15 mM) and leads to complete inhibition of enzyme activity. The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (approximately 39 microM) than when it is reduced. By contrast, binding and release of cyanide are sluggish. The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme. Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme. The biscyano complex is inactive. These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme.     

Look on the positive side! The orientation, identification and bioenergetics of 'Archaeal' membrane-bound nitrate reductases

Many species of Bacteria and Archaea respire nitrate using a molybdenum-dependent membrane-bound respiratory system called Nar. Classically, the 'Bacterial' Nar system is oriented such that nitrate reduction takes place on the inside of this membrane. However, the active site subunit of the 'Archaeal' Nar systems has a twin arginine ('RR') motif, which is a suggestion of translocation to the outside of the cytoplasmic membrane. These 'Archaeal' type of nitrate reductases are part of a group of molybdoenzymes with an 'RR' motif that are predicted to have an aspartate ligand to the molybdenum ion. This group includes selenate reductases and possible sequence signatures are described that serve to distinguish the Nar nitrate reductases from the selenate reductases. The 'RR' sequences of nitrate reductases of Archaea and some that have recently emerged in Bacteria are also considered and it is concluded that there is good evidence for there being both Archaeal and Bacterial examples of Nar-type nitrate reductases with an active site on the outside of the cytoplasmic membrane. Finally, the bioenergetic consequences of nitrate reduction on the outside of the cytoplasmic membrane have been explored.