The copper content of recombinant CotA laccase from Bacillus subtilis produced by Escherichia coli cells is shown to be strongly dependent on the presence of copper and oxygen in the culture media. In copper-supplemented media, a switch from aerobic to microaerobic conditions leads to the synthesis of a recombinant holoenzyme, while the maintenance of aerobic conditions results in the synthesis of a copper-depleted population of proteins. Strikingly, cells grown under microaerobic conditions accumulate up to 80-fold more copper than aerobically grown cells. In vitro copper incorporation into apoenzymes was monitored by optical and electron paramagnetic resonance (EPR) spectroscopy. This analysis reveals that copper incorporation into CotA laccase is a sequential process, with the type 1 copper center being the first to be reconstituted, followed by the type 2 and the type 3 copper centers. The copper reconstitution of holoCotA derivatives depleted in vitro with EDTA results in the complete recovery of the native conformation as monitored by spectroscopic, kinetic and thermal stability analysis. However, the reconstitution of copper to apo forms produced in cultures under aerobic and copper-deficient conditions resulted in incomplete recovery of biochemical properties of the holoenzyme. EPR and resonance Raman data indicate that, presumably, folding in the presence of copper is indispensable for the correct structure of the trinuclear copper-containing site.

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.

Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.

Direct electronic coupling of peroxidases with bio-compatible interfaces allows for investigation of enzyme's electro-catalytic properties that are essential in the design of bio-electronic devices. Here, a novel dye decolourising-type peroxidase from Pseudomonas putida MET94 (PpDyP) is immobilised on Ag electrodes coated with an alkanethiol self-assembled monolayer. Structural features of the active site, heterogeneous electron transfer and electro-catalytic properties of immobilised PpDyP are addressed by combination of surface enhanced spectroscopic and electrochemical approaches. They reveal that the structural integrity of the heme pocket of PpDyP is preserved upon immobilisation, the enzyme is electronically coupled to the electrode, and it exhibits efficient catalytic activity. Importantly, no significant modulation of the midpoint redox potential (E m ) of the immobilised protein (E m À300 mV) is observed with respect to that in solution (E m,sol À260 mV). This study provides important structural and mechanistic insights into immobilised DyP-type peroxidase, capable of efficient decolourisation of numerous dyes, revealing PpDyP as a promising candidate for biotechnological applications.

Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10−7 m and that 2–8% (∼ 2–5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme-binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10−7 m. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme.