Structural Microheterogeneity of a Tryptophan Residue Required for Efficient Biological Electron Transfer between Putidaredoxin and Cytochrome P-450_cam

The carboxy-terminal tryptophan of putidaredoxin, the Fe₂S₂-·Cys₄ iron-sulfur physiological redox partner of cytochrome P-450_cam, is essential for maximal biological activity [Davies, M. D., Qin, L., Beck, J. L., Suslick, K. S., Koga, H., Horiuchi, T., & Sligar, S. G. (1990) J. Am. Chem. Soc. 112, 7396-7398]. This single tryptophan-containing protein thus represents an excellent system for studying the solution dynamics of a residue directly implicated in an electron-transfer pathway. Steady-state and time-resolved measurements of the tryptophan fluorescence have been conducted across the emission spectrum as a function of redox state to probe potential structural changes which might be candidates for structural gating phenomena. The steady-state emission spectrum (λ_max = 358 nm) and anisotropy (α = 0.04) suggest that Trp-106 is very solvent-exposed and rotating partially free of global protein constraints. The time-resolved fluorescence kinetics for both oxidized and reduced putidaredoxin are fit best with three discrete components of approximately 5, 2, and 0.3 ns. The lifetime components were assigned to physical species with iodide ion quenching experiments, where differential quenching of the longer components was observed (k_τ=2 = 5.9 × 10⁸ M^-l s^-1, k_τ=5 = 1.3 × 10⁸ M^-1 s^-1). These findings suggest that the multiexponential fluorescence decay results from ground-state conformational microheterogeneity and thus demonstrate that the essential tryptophan exists in at least two distinguishable conformations. Small differences in the relative proportions of the components between redox states were observed but not cleanly resolved. It thus appears that Trp-106 exists in similar microconformations for both oxidized and reduced putidaredoxin, characterized by three lifetimes with similar relative proportions. These results do not rule out differences within the cytochrome P-450_cam complex and indeed suggest potential mechanisms for selection of specific geometries. The absence of fluorescence lifetime wavelength dependencies demonstrates that each species displays the red-shifted, solvent-exposed, steady-state spectra. Differential solvent exposure is thus unlikely to be the primary mechanism for the asymmetric iodide quenching. Differential interactions of the tryptophan microconformations with an anionic protein surface are proposed as a mechanism for the iodide effects and supported by quenching studies with the neutral quenching agent acrylamide. The mechanism is consistent with a physical model of putidaredoxin-cytochrome P-450_cam complex formation where the negatively charged surface is suggested to be the anionic binding surface of putidaredoxin implicated in putidaredoxin reductase and cytochrome P-450_cam interactions [Stayton, P. S., & Sligar, S. G. (1990) Biochemistry 29, 7381-7386]. This model thus places the essential Trp-106 directly at the cytochrome P-450_cam binding interface in position to mediate electron transfer.