The stability and reproducibility of most SERS-active electrode surfaces are far from ideal. We have focused on this problem by developing and characterizing a metal film over nanosphere (MFON) electrode which solves these shortcomings. Atomic force microscopy (AFM), cyclic voltammetry, and surface-enhanced Raman spectroscopy (SERS) of representative molecules were used to characterize and evaluate the electrochemical and SERS performance of MFON electrodes. Tremendous stability to extremely negative potential excursions is observed for MFON electrodes as compared to standard metal oxidation reduction cycle (MORC) roughened electrodes. Consequently, irreversible loss of SERS intensity at negative potentials is not observed on these MFON electrodes. We conclude that MFON electrodes present a significant advantage over MORC electrodes because SERS enhancement is not lost upon excursion to extremely negative potentials. This work demonstrates that the MFON substrate, while easily prepared and temporally stable, offers unprecedented stability and reproducibility for electrochemical SERS experiments. Furthermore, one can conclude that irreversible loss is not a distinguishing characteristic of electrochemical SERS and consequently cannot be used as evidence to support the chemical enhancement mechanism.

The distance and orientation dependence of the heterogeneous electron-transfer reaction between ferrocytochrome c (Fe 2+ Cc) and a silver film over nanosphere (AgFON) electrode is examined in detail using electrochemical surface-enhanced resonance Raman spectroscopy (SERRS) as a molecularly specific and structurally sensitive probe. The distance between the Fe 2+ redox center and the electrode surface is controlled by varying the chain length x of an intervening carboxylic acid terminated alkanethiol, HS(CH 2 ) x COOH, self-assembled monolayer (SAM). The orientation of the heme in Fe 2+ Cc with respect to the AgFON/S(CH 2 ) x -COOH electrode surface is controlled by its binding motif. Electrostatic binding of Fe 2+ Cc to AgFON/S(CH 2 ) x -COOH yields a highly oriented redox system with the heme edge directed toward the electrode surface. The binding constants were determined to be K ) 5.0 × 10 6 M -1 and 1.1 × 10 6 M -1 , respectively, for the x ) 5 and x ) 10 SAMs. In contrast, covalent binding of Fe 2+ Cc yields a randomly oriented redox system with no preferred direction between the heme edge and the electrode surface. SERRS detected electrochemistry demonstrates that Fe 2+ Cc electrostatically bound to the x ) 5 AgFON/S(CH 2 ) x COOH surface exhibits reversible oxidation to ferricytochrome c, whereas Fe 2+ Cc electrostatically bound to the x ) 10 surface exhibits irreversible oxidation. In comparison, Fe 2+ Cc covalently bound to the x ) 5 and x ) 10 surfaces both exhibit oxidation with an intermediate degree of reversibility. In addition to these primary results, the work presented here shows that AgFON/S(CH 2 ) x COOH surfaces (1) are biocompatible -Fe 2+ Cc is observed in its native state and (2) are stable to supporting electrolyte changes spanning a wide range of ionic strength and pH thus enabling, for the first time, SERRS studies of these variables in a manner not accessible with either the widely used colloid or electrochemically roughened SERS-active surfaces.

We report that when mixed-metal, [M, Fe] hemoglobin (Hb) hybrids, with Fe in one type of subunit and M = Zn or Mg in the other type, are embedded in clear poly(vinyl alcohol) (PVA) films, they exhibit inter-subunit electron transfer (ET) electron−nuclear tunneling down to cryogenic temperatures (5 K), making them the first protein system other than photosynthetic systems to exhibit such behavior. The rate constant for the (Fe2+Porphyrin) → (MPorphyrin)+ inter-subunit ET reaction shows a roughly temperature-invariant, quantum-tunneling regime from cryogenic temperatures (5 K) up to ca. 200 K. Some of the hybrids (depending on M and the Fe ligand) begin to show a strong increase in this ET rate constant at higher temperatures. This behavior is discussed here in terms of a recent heuristic description of ET in a glassy environment that accounts for the fact that slow solvent relaxation at low temperatures, and in particular upon cooling through a glassing transition, causes the reaction pathway to deviate from the path through the equilibrium transition state, and leads to the formation of nonequilibrium ET product states represented by points on the product surface other than that of the equilibrium product state. The analysis suggests that in regard to the dynamical modes of motion that control ET, the protein “medium” acts substantially like a frozen glass, even at room temperature. It further suggests that, although the protein acts largely as its own heat bath, the thermal characteristics of that heat bath can be modified by the external environment.

The Tyr alpha 42-Asp beta 99 intersubunit H-bond stabilizes the T quaternary structure in hemoglobin (Hb) tetramers. We had proposed that Tyr alpha 42 acts as an acceptor in this H-bond, because the tyrosine Y8a/8b and Y7a' UVRR (ultraviolet resonance Raman) bands shift in directions opposite to those expected if tyrosine is an H-bond donor. If Asp beta 99 is the H-bond donor, then it must be protonated in the T state, and would be a previously unrecognized contributor to the Bohr effect. This implication was strengthened by the discovery that an R-minus-T difference FTIR (Fourier transform infrared) band at 1693 cm-1, which might be a signal from protonated carboxylate, is missing in Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn. However, we now find that this FTIR signal is insensitive to 13C-labeling of the aspartate residues in Hb, and cannot arise from protonated Asp beta 99. There are no other difference signals in the 1700 cm-1 region at a sensitivity of one COOH group. We conclude that Asp beta 99 is not protonated, and that the anomalous UVRR shifts must arise from compensating polarization of the Tyr alpha 42 OH. Candidates for this compensation are the H-bond donated by the Asp beta 94 backbone NH, and the nearby positive charge of Arg beta 40.

UV resonance Raman difference spectra between ligated and deoxyhemoglobin contain tryptophan and tyrosine signals which arise from quaternary H-bonds in the T state, which are broken in the R state. These H-bonds are unaffected by bis(3,5-dibromosalicyl) fumarate cross-linking at the Lys alpha 99 residues, which prevents dissociation of Hb tetramers to dimers. However, when the pH is lowered from 9.0, or when NaCl is added, intensity is diminished for the tyrosine Y8 and tryptophan W3 bands of cross-linked deoxyHb, but not of native deoxyHb. This effect is attributed to weakening of tertiary H-bonds involving Tyr alpha 140 and Trp alpha 14, when the T state salt bridge between Val alpha 1 and Arg alpha 141 is formed via protonation of the terminal amino group and anion binding. The Tyr alpha 140-Val alpha 93 H-bond connects the Arg alpha 141-bearing H helix with the Lys alpha 99-bearing G helix. Weakening of the H-bond reflects a tension between the fumarate linker and the salt-bridge. This tension inhibits protonation of the Val alpha 1 amino terminus, thus accounting for the diminution of both proton [Bohr effect] and CO2 binding in the T state as a result of cross-linking.