Re(bpy)(CO)3CN as a probe of conformational flexibility in a photochemical ribonucleotide reductase.
ABSTRACT Photochemical ribonucleotide reductases (photoRNRs) have been developed to study the proton-coupled electron transfer (PCET) mechanism of radical transport in Escherichia coli class I ribonucleotide reductase (RNR). The transport of the effective radical occurs along several conserved aromatic residues across two subunits: beta2((*)Y122 --> W48 --> Y356) --> alpha2(Y731 --> Y730 --> C439). The current model for RNR activity suggests that radical transport is strongly controlled by conformational gating. The C-terminal tail peptide (Y-betaC19) of beta2 is the binding determinant of beta2 to alpha2 and contains the redox active Y356 residue. A photoRNR has been generated synthetically by appending a Re(bpy)(CO)(3)CN ([Re]) photo-oxidant next to Y356 of the 20-mer peptide. Emission from the [Re] center dramatically increases upon peptide binding, serving as a probe for conformational dynamics and the protonation state of Y356. The diffusion coefficient of [Re]-Y-betaC19 has been measured (k(d1) = 6.1 x 10(-7) cm(-1) s(-1)), along with the dissociation rate constant for the [Re]-Y-betaC19-alpha2 complex (7000 s(-1) > k(off) > 400 s(-1)). Results from detailed time-resolved emission and absorption spectroscopy reveal biexponential kinetics, suggesting a large degree of conformational flexibility in the [Re]-Y-betaC19-alpha2 complex that engenders partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions.
Article: Photoactive peptides for light-initiated tyrosyl radical generation and transport into ribonucleotide reductase.[show abstract] [hide abstract]
ABSTRACT: The mechanism of radical transport in the alpha2 (R1) subunit of class I E. coli ribonucleotide reductase (RNR) has been investigated by the phototriggered generation of a tyrosyl radical, *Y356, on a 20-mer peptide bound to alpha2. This peptide, Y-R2C19, is identical to the C-terminal peptide tail of the beta2 (R2) subunit and is a known competitive inhibitor of binding of the native beta2 protein to alpha2. *Y356 radical initiation is prompted by excitation (lambda >or= 300 nm) of a proximal anthraquinone, Anq, or benzophenone, BPA, chromophore on the peptide. Transient absorption spectroscopy has been employed to kinetically characterize the radical-producing step by time resolving the semiquinone anion (Anq*-), ketyl radical (*-BPA), and Y* photoproducts on (i) BPA-Y and Anq-Y dipeptides and (ii) BPA/Anq-Y-R2C19 peptides. Light-initiated, single-turnover assays have been carried out with the peptide/alpha2 complex in the presence of [14C]-labeled cytidine 5'-diphosphate substrate and ATP allosteric effector. We show that both the Anq- and BPA-containing peptides are competent in deoxycytidine diphosphate formation and turnover occurs via Y731 to Y730 to C439 pathway-dependent radical transport in alpha2. Experiments with the Y730F mutant exclude a direct superexchange mechanism between C439 and Y731 and are consistent with a PCET model for radical transport in which there is a unidirectional transport of the electron and proton transport among residues of alpha2.Journal of the American Chemical Society 07/2007; 129(27):8500-9. · 9.91 Impact Factor
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ABSTRACT: The complex Re(bpy)(CO)3CN is an excited state oxidant of tyrosine upon deprotonation of the tyrosyl phenol. A series of Re(bpy-FnY)(CO)3CN complexes ([Re]-FnY: [Re]-Y, [Re]-3-FY, [Re]-3,5-F2Y, [Re]-2,3-F2Y, [Re]-2,3,5-F3Y, [Re]-2,3,6-F3Y, and [Re]-F4Y) have been prepared so as to vary the FnY*/FnY- reduction potential and thus the driving force for electron transfer in this system. Time-resolved emission and nanosecond absorption spectroscopies have been used to measure the rates for charge separation, CS, and charge recombination, CR, for each complex. A driving force analysis reveals that CS is well described by Marcus' theory for ET, is strongly driving force dependent (activated), and occurs in the normal region for ET. CR, on the other hand, is weakly driving force dependent (near activationless) and occurs in the inverted region for ET. These data demonstrate that fluorotyrosines will be powerful probes for unraveling charge transport mechanisms in enzymes that utilize tyrosyl radicals.Journal of the American Chemical Society 11/2006; 128(42):13654-5. · 9.91 Impact Factor
Chemical Communications 11/2003; · 6.17 Impact Factor