No abstract

The blue color in metalloprotein azurin has traditionally been attributed to the intense cysteine-to-Cu2+ ligand-to-metal charge transfer transition centered at 628 nm. Although resonance Raman measurements of the Cu2+ active site have implied that the LMCT transition electronically couples to the protein scaffold well beyond its primary metal–ligand coordination shell, the structural extent of this electronic coupling and visualization of the protein-mediated charge transfer dynamics have remained elusive. Here, using femtosecond broadband transient absorption and impulsive Raman spectroscopy, we provide direct evidence for a rapid relaxation between two distinct charge transfer states, having different spatial delocalization, within ∼300 fs followed by recombination of charges in subpicosecond time scales. We invoke the formation of a protein-centered radical cation, possibly Trp48 or a Phe residue, within 100 fs substantiating the long-range electronic coupling for the first time beyond the traditional copper active site. The Raman spectra of the excited CT state show the presence of protein-centric vibrations along with the vibrational modes assigned to the copper active site. Our results demonstrate a large delocalization length scale of the initially populated CT state, thereby highlighting the possibility of exploiting azurin photochemistry for energy conversion techniques.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding.Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metalbound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by $100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.