Rational design of a structural and functional nitric oxide reductase

Abstract

Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally, it is more difficult to design functional proteins. In comparison to recent successes in designing non-metalloproteins, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem Fe(B) centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

Figure 1

Crystal structure of rationally designed Fe_BMb overlays closely with minimized computer model. A) Minimized computer model of Fe_BMb with Zn(II) in the Fe_B site. B) Crystal structure of Fe(II)-Fe_BMb collected at Fe-edge absorption (1.7309 Å) at the Brookhaven National Synchrotron Light Source X12C beamline (Upton, NY) with 1.72 Å resolution. The Fe•••Fe distance is 4.82 Å, while the Fe-O-Fe angle is 115° (OE1 atom of E68). C) Overlay of Fe_BMb model (yellow) with Fe(II)-Fe_BMb crystal structure (cyan). In general, Fe(II) of the Fe_B site is represented by a green sphere; Zn(II) (grey sphere) was used to model the Fe_B site. A water molecule in the heme pocket is represented by a red sphere.

Figure 2

Designed Fe_BMb has NOR activity in the presence of Fe²⁺ and NO. Control proteins show no evidence of NO activity under identical conditions. A) UV-vis spectra of 10 μM of deoxy Fe_BMb (a), deoxy wtMb (b), and deoxy Fe_BMb(-Glu) (c) in the absence (black spectra) or presence (red spectra) of 2 eq Fe²⁺. B) UV-vis spectra of the NO reaction of 10 μM of deoxy Fe_BMb (a), deoxy wtMb (b), and deoxy Fe_BMb(-Glu) (c) in the absence (black spectra) or presence (red spectra) of 2 eq Fe²⁺ after 20 min of incubation with ~17 eq NO. The deoxy protein was formed by reaction of met protein with excess dithionite in a glove box. Gel filtration purification was used to remove excess dithionite to preclude its reaction with NO.

Figure 3

Product of NO reaction by Fe_BMb is N₂O. Time dependent GC/MS measurements of N₂O formation by Fe(II)-Fe_BMb (A). Control reactions of NO with Fe²⁺ only (B) and wtMb (C). NO (~17 eq) was reacted with Fe(II)-Fe_BMb (0.6 mM protein, 1.5 mM or 2.5 eq Fe²⁺), Fe²⁺ (1.5 mM, no protein), or with wtMb (1 mM) and Fe²⁺ (2 mM or 2 eq). At 6 hr, 2 eq dithionite (1.2 mM) was added and allowed to react to simulate a second turnover. NO₂ (MW 46) was not detected. GC peaks have been normalized. N₂O yield (30%) was determined from a comparison of the ratio of the NO:N₂O peaks of the 30 MW:44 MW single ion chromatograms 2 hr after dithionite addition (after which additional N₂O formation was not observed), to that of known dithionite concentrations (2 hr after addition). Background N₂O formation (i.e., Fe²⁺ without protein) was subtracted from that of Fe(II)-Fe_BMb to estimate the yield.