A FRET Enzyme-Based Probe for Monitoring Hydrogen Sulfide Maria Strianese,* ,† Gottfried J. Palm,* ,‡ Stefano Milione, † Olaf Kü hl, ‡ Winfried Hinrichs, ‡ and Claudio Pellecchia † † Dipartimento di Chimica e Biologia, Universita ̀ degli Studi di Salerno, via Ponte Don Melillo, I-84084 Fisciano (Sa), Italy ‡ Institute for Biochemistry, University of Greifswald, Felix-Hausdorff Strasse 4, 17489 Greifswald, Germany * S Supporting Information ABSTRACT: Fluorescently labeled cobalt peptide defor- mylase (Co-PDF) can be efficiently used as a fluorescence- resonance-energy-transfer-based sensing device for hydro- gen sulfide (H 2 S). The proof of concept of our sensor system is substantiated by spectroscopic, structural, and theoretical results. Monohydrogen sulfide coordination to Co-PDF and Ni-PDF was verified by X-ray crystallog- raphy. Density functional theory calculations were performed to gain insight into the characteristics of the coordination adduct between H 2 S and the cobalt cofactor in Co-PDF. H ydrogen sulfide (H 2 S) is among the oldest and simplest of molecules whose reaction chemistry has very recently attracted attention from several research groups. 1 For hundreds of years, it has been known solely as a harmful gas. 2 More recently, H 2 S has emerged as the “third gaseous transmitter” in biology, along with nitric oxide and carbon monoxide. 3,4 Selective tracking of this small molecule in physiological conditions is particularly relevant to elucidate its complex contributions to both healthy and disease states. The challenge calls for a new generation of biocompatible sensing devices to assess endogenous concentrations of H 2 S. 5 Very recently, fluorescence-based systems for H 2 S detection have been proposed as effective probes for biological applications. 5-8 These probes can detect H 2 S in aqueous solution with high sensitivity and selectivity. 5-7 In all of these systems, the molecule acting as the recognition element has an abiotic origin. Aiming to develop more biocompatible devices, we focused on the design of fluorescence-enzyme-based biosensors. It is well-known that H 2 S can bind to heme proteins, inducing different responses that, in turn, modulate its cytotoxic and cytoprotective activities. 9 Thus, the first system that we devised as a fluorescent H 2 S sensor makes use of a heme protein. In particular, we employed myoglobin from horse skeletal muscle (Mb). 10 A limitation of our Mb monitoring system is the low amplitude of the fluorescence signals. Furthermore, by subsequent additions of H 2 S, Mb(Fe 3+ ) is reduced to the ferrous form: the cuvette sample resulted in a mixture of Mb(Fe 3+ )-H 2 S, Mb(Fe 2+ )-H 2 S, and Mb(Fe 2+ ). A similar reduction has been observed by Scheidt et al. for monohydrogen sulfide (HS - ) coordination in iron porphyrinates. 11 To improve the Mb-based H 2 S sensor, we had to overcome some of its limitations. In the current work, we extended the same approach to cobalt-containing peptide deformylase from Escherichia coli (Co-PDF). PDF’s native metal cofactor is Fe II , but it can easily be replaced by other thiophilic metal ions. Whereas PDF with Fe II (Fe-PDF) is very sensitive to molecular oxygen, the variants Co-PDF, Ni-PDF, and Zn-PDF are stable. 12,13 PDF has the same overall structure with any of the four metals. 14 The active site metal is coordinated by the imidazole atoms His132Nε2 and H136Nε2 and the thiolate C90Sγ with trigonal-pyramidal geometry. In the resting state, one water molecule (possibly two water molecules at low pH) completes the metal coordination polyhedron. The Glu133 side chain polarizes or deprotonates one aqua ligand during formyl peptide hydrolysis. Cobalt is known to have a strong affinity for sulfur-containing ligands, as does nickel. 15,16 In the first instance, we focused on Co-PDF for our experiments. To test the principal usability of the system as an optical H 2 S biosensor, H 2 S binding to Co-PDF was assessed via UV-visible spectroscopy. When H 2 S was bubbled through a Co-PDF solution, the absorption spectrum signi ficantly changed. Specifically, while the absorption spectrum of Co-PDF exhibits a band at 280 nm and three less intense bands centered at 320, 560, and 660 nm, 17 H 2 S addition quenches the 560 and 660 nm bands and leads to the appearance of two new bands at 625 and 665 nm (Figure 1A and Figure S1 in the Supporting Information, SI). Spectroscopic features of Co-PDF make it ideally suited for implementing a fl uorescence-resonance-energy-transfer Received: June 26, 2012 Published: October 16, 2012 Figure 1. (A) Absorption spectrum of H 2 S-free Co-PDF (gray) and the H 2 S-bound form (dark gray) and emission spectrum (dotted trace) of Atto620 (λ max = 645 nm). Protein concentration: 125 μM in a 100 mM potassium phosphate buffer (pH = 6.8, room temperature). (B) Fluorescence emission spectrum (λ ex = 620 nm) of Atto620-labeled H 2 S-free (black trace) and H 2 S-bound (gray trace) Co-PDF. Protein concentration: 100 nM. In this experiment, Co-PDF was labeled on the amines. Communication pubs.acs.org/IC © 2012 American Chemical Society 11220 dx.doi.org/10.1021/ic301363d | Inorg. Chem. 2012, 51, 11220-11222