I would like to thank all my friends in the Chemistry Department, as well as many others outside the Chemistry Department, who have been a big part of my social life. In the cases where the wire was shorter, ligation of the Fe(II) species was not observed.
ELECTRON TRANSFER THROUGH PROTEINS
ELECTRON TRANSFER THEORY
In the absence of interactions with other species (such as quenchers), the energy transfer will be followed by radiative or nonradiative decay of the excited acceptor state. Our studies show that a large part of the rearrangement energy and electron transfer rates depend on the nuclear reorientation of the protein.
WIRES PROJECT
The inorganic nature of the metal complex enabled easy crystallization of the thread-protein complexes, revealing interesting protein-substrate conformations. All the rhenium-based leads are active site channel binders of iNOS, characterized by UV-Vis spectroscopy.
NITRIC OXIDE SYNTHASE
Docking of the oxygenase domain on top of the reductase domain (PBD code 1MMV and 1TLL). The arrow indicates the flip direction of the FMN domain and the circle indicates the hydrophobic patch of the oxygenase domain that interacts with the FMN domain.
The catalytic mechanism of mammalian inducible nitric oxide synthase (iNOS) was investigated with a sensitizer (wire)-linked substrate to characterize the intermediates formed by laser-induced electron transfer to the active site of the enzyme. The binding of the nitro group to the Fe heme could be mechanistically analogous to the N-oxy binding of N-hydroxyarginine during the second turn of the catalytic cycle.
INTRODUCTION
The rhenium metal complex was designed to be small enough to easily access the opening of the iNOS channel. The nitroarginine substrate was introduced at the wire terminus for mechanistic studies on the turnover of electron transfer processes induced by photoexcitation of the rhenium center.
EXPERIMENTALS
A saturation binding experiment measures the equilibrium binding of the inhibitor ([L]) at various concentrations to determine the number of binding sites (Bmax) and the ligand affinity (Kd). The amplitudes of the decay traces were used to determine the ratio of bound wire to free wire (cn/cn+1).
RESULTS
When the wire is bound to iNOSoxy, the excited state Re(I) emission (Re(I)*) will decrease compared to the Re(I)* emission intensity of the wire in buffer (Figure 2.6). From the transient absorption data, Re(I)* has an absorption decay of ~ 580 ns, which is close to the Re(I)* luminescence decay trace (Figure 2.12). The instantaneous appearance of the Fe(II) species indicates that Fe(II) is formed in less than 10 ns (instrument response limit), which is an order of magnitude faster than the naturally occurring Fe(II) species produced under biological conditions (kET = 1 s -1).6,21.
Single-wavelength transient absorption traces of different concentrations of ReC8argNO2 in the protein are shown in Figure 2.15.
DISCUSSIONS
Raman transient absorption and resonance data reveal that the Fe(II) species has a sixth ligand similar to NO. The wire has a nitro-terminated arginine substrate that is electronically similar to NO and interacts closely with the Fe-heme. After photoreduction of the Fe-heme, it is proposed that the nitro group itself ligates the Fe(II)-heme, creating a low-spin, six-coordinate Fe(II) species detectable by transient absorption and resonance Raman spectroscopy ( Scheme 2.5).
Upon binding of ReC8argNO2 to iNOSoxy, the Fe heme switches from low-spin Fe(III) to Fe(III) (step 1).
CONCLUDING REMARKS
Two sensitizer-coupled substrates (wires), ReC3arg and ReC3argNO2, were synthesized to investigate the catalytic cycle of iNOSoxy. The wires were based on the structure and composition of ReC8argNO2 from Chapter II. The Re(I)* excited states of both wires were reductively quenched by ascorbate and para-methoxy-N,N'-dimethylaniline (pMDA).
Re(0) was generated after reductive quenching and subsequently reduced Fe(III) to Fe(II) in less than 10 ns.
INTRODUCTION
EXPERIMENTALS
The solution was concentrated by rotary evaporation and purified by flash column chromatography (10:1 CH 2 Cl 2 :MeOH) to collect a yellow oil product (920 mg, 92% yield). The reaction mixture was concentrated by rotary evaporation and purified by flash column chromatography (CH 2 Cl 2 was used as the first eluent and the final eluent was 50:3 CH 2 Cl 2 :MeOH). The solution was concentrated by rotary evaporation and purified by flash column chromatography (10:1 CH 2 Cl 2 :MeOH).
The product was purified by flash column chromatography (1:1 ACN:H2O) and collected as a yellow oil (440 mg, 100% yield).
BINDING RESULTS FOR REC 3 ARG
At a 3:1 equivalent concentration of wire to protein (third solid line from bottom of Figure 3.8), the Re(I)* fluorescence intensity overlaps the Re(I)* intensity of two equivalents of wire in buffer (e the second dotted line from the bottom of figure 3.8). Assuming that the bound wire does not fluoresce, it is concluded that at a 3:1 concentration of wire to protein, two equivalents of wire are free in solution, while one equivalent of wire is bound to the protein. The location of the second binding site could not be inferred from the steady-state fluorescence data.
This suggested that slightly more than one equivalent of wire is bound to the protein, which is consistent with steady-state fluorescence.
BINDING RESULTS FOR REC 3 ARGNO 2
UV-Vis spectroscopy of ReC3argNO2 titers in the iNOSoxy sample; difference spectra are shown as insets. This was evident with small spectral changes observed for the titration of ReC3argNO2 in iNOSoxy samples. Steady-state fluorescence traces of ReC3argNO2 titrations in iNOSoxy (solid lines) and buffer (dotted lines) samples.
Transient lifetime decay traces of ReC3argNO2 in buffer (dashed lines) and in the presence of iNOSoxy (solid lines).
ELECTRON TRANSFER KINETICS (PART I): RESULTS USING 1 CM PATH LENGTH CUVETTE
Transient absorption trace of thread in the presence of protein is shown in Figure 3.24 as the blue trace. Transient absorption curve of ReC3argNO2 in the presence of pMDA plotted at 2 μs (circles) and 4 μs (stars). The intensity of the long-lived signal in the presence of protein (green dotted trace) is reduced compared to the trace without protein (orange dotted trace).
Transient absorption curve of ReC3argNO2 in the presence of pMDA (dashed triangle line) and in the presence of pMDA and iNOSoxy (solid traces) plotted after 2 μs (stars), 3 μs (rectangles) and 4 μs (circles) to avoid Re (0) absorbance interferences .
ELECTRON TRANSFER KINETICS (PART II): RESULTS USING 0.5CM PATH LENGTH CUVETTE
Transient absorbance curve of ReC3argNO2 and ascorbate in the absence of iNOSoxy (open circles) and in the presence of iNOSoxy (closed shapes) after 2 μs (diamonds), 3 μs (asterisks) and 4 μs (circles). Transient absorbance curve for ReC3arg and ascorbate in the absence of iNOSoxy (open circles) and in the presence of iNOSoxy (closed shapes) after 2 μs (diamonds), 3 μs (asterisks) and 4 μs (circles). Transient absorbance curve for ReC3argNO2 and pMDA in the absence of iNOSoxy (open circles) and in the presence of iNOSoxy (closed shapes) after 2 μs (circles), 3 μs (diamonds), and 4 μs (asterisks).
Transient absorption curve of ReC3argNO2 and pMDA in the absence of iNOSoxy (open circles) and in the presence of iNOSoxy (closed shapes) after 2 μs (circles), 3 μs (diamonds), and 4 μs (stars).
DISCUSSIONS
(+)Trp recombines with the Fe(II) species and the electron transfer cycle is complete. A direct electron transfer from the Re(I)* state was completed, impossible due to a small difference in redox potential between substrate and product (step 6). Upon binding of ReC3argNO2, Fe(III) loses the water ligand and becomes a five-coordinate, high-spin Fe(III) heme.
The (+) quencher recombines with the Fe(II) species and the electron transfer cycle is complete.
CONCLUDING REMARKS
Ru(dmbpy)(aminophenanthroline-C10F9)][PF6]2 (RuphenF9bp) binds to the protein surface with a Kd of 500 ± 60 nM, presumably at the rear of the channel where the FMN domain is expected to occupy the full-length enzyme complex during electron transfer processes. It is proposed that the electron travels along a similar pathway to the natural electron transfer pathway from the FMN domain.
INTRODUCTION
A fourth ruthenium wire was designed and synthesized for binding and inhibition studies with the oxygenase domain of full-length iNOS (iNOSoxy) to serve as a comparison to the three existing ruthenium wires (Figure 4.3, RuphenF9bp) examined with Δ65 and Δ114 iNOSoxy. The synthetic step, from the primary amine nucleophilic attack to the para-carbon sites, provides a better and cleaner coupling reaction than what was used for previous wires. The bulky ruthenium center and electrostatic properties of the decafluorophenanthroline ligand are retained to promote surface binding effects.
Surface-binding ruthenium wires may provide an effective means of inhibiting NOS by preventing electron transfer between the reductase and oxygenase domains.
EXPERIMENTALS
Then, 1 ml (22 mmol) of hydrazine monohydrate was added dropwise to the reaction mixture and heated to 65oC. The filtrate was concentrated by rotary evaporation and carried to the next step without further purification. The solution was refluxed at 78oC for 1 hour until the reaction mixture turned purple.
The volume was reduced and the precipitate was collected by vacuum filtration.
RESULTS
Initially, the emission intensity of the wire in the protein complex was enhanced compared to that of the wire in the buffer. The transient absorbance of RuphenF9bp in the presence of ascorbate and TMPD was measured at different wavelengths (nm) (Figure 4.19). The transient absorbance of the wire in the presence and absence of the suppressor was overlaid for comparison (Figure 4.19, dotted trace).
Transient absorbance was measured for the wire in the presence of ascorbate, TMPD and iNOSoxy (Figure 4.21).
DISCUSSIONS
However, the back of the channel is more open for the thread to bind closer to the Fe heme at 4.91 Å away (Figure 4.31, orange RuphenF9bp). The back binding site is most likely the preferred binding site for RuphenF9bp, due to its easy access and proximity to the Fe heme. Another possible ET pathway would be a one-step tunnel from the ruthenium center to the Fe heme (distance = 17 Å).
A hopping mechanism may dominate from the ruthenium center to Lsy423,Trp409 and then to the Fe heme, a total distance of 24.6 Å, which is the proposed ET pathway from the reductase domain to the Fe heme.16.
CONCLUDING REMARKS
