We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

The reduction of fluid lecithin bilayer-substrate coupling by the presence of a hydrophilic polymer cushion in spherical supported bilayers of dipalmitoylphosphatidylcholine (DPPC) has been studied by measuring the lecithin molecular order and lateral diffusion using a deuterium NMR relaxation method. We obtained for both monolayers of the supported bilayer a lateral diffusion coefficient (D ) 13.5 × 10 -12 m 2 /s at 55 °C) similar to that measured previously in the outer monolayer of a supported bilayer on a bare silica surface, indicating that the presence of the polymer cushion can indeed largely eliminate the slow down effect of the solid surface on the bilayer dynamics. Moreover, we found that the polymer cushion rendered the molecular order of the lipids identical to that measured for multilamellar bilayer vesicles (MLV). A comparison of the measured D values with those obtained for oriented planar multilayer stacks of DPPC strongly suggests that lateral diffusion in these systems is still a factor of 2 faster than that of single bilayers on a support.

The molecular order and lateral diffusion in fluid cationic bilayers of chain perdeuterated DPTAP (1,2dipalmitoyl-d62-trimethylammonium-propane) on a spherical solid support were studied by deuterium NMR and microcalorimetry. In comparison to the zwitterionic perdeuterated DPPC (1,2-dipalmitoyl-d62sn-glycero-3-phosphocholine), the molecular order parameter profile of the palmitoyl chains of DPTAP shows significantly higher molecular order toward the interface while similar values were obtained in the vicinity of the bilayer center. The lateral diffusion coefficient D of DPTAP is D ) (13.2 ( 1.5) × 10 -12 m 2 /s at 60 °C, slightly below that of DPPC at this temperature. Increasing the ionic strength caused the value of D to decrease further. The results suggest that despite the cationic charge the supported DPTAP bilayer features tighter molecular packing and a reduced lateral diffusivity of its constituents than for the zwitterionic DPPC on a solid support.

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