Redox-dependent super(1)H NMR spectral features and tertiary structural constraints on the C-terminal region of putidaredoxin

• Published in: Biochemistry (Easton) Vol. 33; no. 21; pp. 6433 - 6441
• Main Authors: Pochapsky, T C, Ratnaswamy, G, Patera, A
• Format: Journal Article
• Language: English
• Published: 01.01.1994
• ISSN: 0006-2960

Putidaredoxin (Pdx) is a 106-residue Fe sub(2)S sub(2) ferredoxin which acts as the physiological reductant and effector of cytochrome P-450 sub(cam). Pdx has two accessible oxidation states, Fe super(+3)-Fe super(3+) (oxidized) and Fe super(+3)-Fe super(+2) (reduced), and exhibits redox-dependent binding affinities for cytochrome P-450 sub(cam), with reduced Pdx binding over 100-fold more tightly than oxidized Pdx to the oxidized cytochrome P-450 sub(cam). The analysis of two-dimensional super(1)H NMR experiments has yielded sequential super(1)H resonance assignments for the diamagnetic regions of the reduced form of Pdx, which are compared to those of oxidized Pdx, described previously. Increased unpaired electron-spin density on the metal cluster in reduced relative to oxidized Pdx increases the number of super(1)H resonances which are broadened by the metal cluster, and the pattern of paramagnetic broadening provides information concerning the placement of the metal cluster within the protein. Two-dimensional exchange experiments on half-reduced samples of Pdx indicate that electron self-exchange is slow on the chemical shift time scale, with a second-order rate constant less than or equal to 66/M/s at 290 K. Spectral changes unrelated to increases in unpaired electron-spin density are also observed. The largest changes of this type are observed for features structurally contiguous with the C-terminal region Pro 102-Trp 106. The C-terminal residue Trp 106 has been implicated in binding to cytochrome P-450 sub(cam). NOE constraints on the structure of this region are described, and a model for the solution structure of Pdx is used to rationalize the observed paramagnetic broadening patterns. A hypothesis is offered to rationalize observed redox-state-dependent binding of Pdx to P-450 sub(cam) and changes in midpoint potential of oxidized Pdx upon binding to oxidized P-450 sub(cam).