Article

Re(bpy)(CO)3CN as a probe of conformational flexibility in a photochemical ribonucleotide reductase.

Department of Chemistry, Massachusetts Institute of Technology,77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, USA.
Biochemistry (impact factor: 3.42). 04/2009; 48(25):5832-8. DOI:10.1021/bi9005804
Source: PubMed

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.

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Keywords

20-mer peptide
 
[Re] center
 
[Re]-Y-betaC19-alpha2 complex
 
biexponential kinetics
 
binding determinant
 
C-terminal tail peptide
 
conformational dynamics
 
conformational flexibility
 
conformational gating
 
conserved aromatic residues
 
effective radical
 
engenders partitioning
 
peptide binding
 
Photochemical ribonucleotide reductases
 
proton-coupled electron transfer
 
protonation state
 
radical transport
 
redox active Y356 residue
 
synthetically
 
time-resolved emission