Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) of ω-carboxylalkanethiols that were covalently attached to Ag electrodes. Employing surface-enhanced resonance Raman (SERR) spectroscopy, the redox equilibria and the structural changes of the adsorbed Cyt-c were analyzed quantitatively for SAMs of different chain lengths ranging from 2-mercaptoacetic acid (C 2 -SAM) to 16mercaptohexadecanoic acid (C 16 -SAM). In the presence of Cyt-c in the bulk solution, the SERR spectra of the adsorbed Cyt-c display the characteristic vibrational band pattern of the native protein conformation denoted as state B1. The enhancement of the SERR signals decreases with increasing chain length, but even at distances as large as 24 Å (C 16 -SAM), SERR spectra of high quality could be obtained. Conversely, no SERR signals could be detected for SAMs including hydroxyl instead of carboxylate headgroups, implying that Cyt-c is adsorbed via electrostatic interactions. On the basis of potential-dependent SERR experiments, the redox equilibria of the adsorbed Cyt-c (B1) were analyzed, revealing ideal Nernstian behavior (n = 1). However, the redox potentials exhibit negative shifts compared to that of Cyt-c in solution, which increase with the chain length of the SAMs. In the absence of excess Cyt-c in solution (i.e., 0.2 µM), a new conformational state B2 of the adsorbed Cyt-c is observed. This state B2, which differs from the native state B1 by the heme pocket structure, includes three substates of different spin and coordination configurations. The distribution among these substates as well as the total contribution of state B2 varies with the chain length of the SAM such that the latter decreases from 73% at C 2 -SAM to 0% at C 11 -and C 16 -SAMs. These results imply that the formation of B2 is induced by the electric field at the binding site, generated by the potential drop across the electrode/SAM interface. When an electrostatic model for the interfacial potential distribution for the electrode/ SAM/protein device is employed, both the redox potential shifts and the electric-field-induced structural changes can be consistently explained. The impact of these findings for the processes of Cyt-c at biological interfaces is discussed.

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IX␣ (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-R c -like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.The phytochrome (Phy) 5 superfamily encompasses a large and diverse set of photoreceptors present in the plant, fungal, and bacterial kingdoms where they play critical roles in various light-regulated processes (1-3). These processes range from the control of phototaxis, pigmentation, and photosynthetic potential in proteobacteria and cyanobacteria to seed germination, chloroplast development, shade avoidance, and flowering time in higher plants. Phys are unique among photoreceptors in being able to assume two stable, photointerconvertible conformers, designated Pr and Pfr based on their respective absorption maxima in the red and far-red spectral regions. By cycling between Pr and Pfr, Phys act as light-regulated switches in various photosensory cascades.Phys are homodimeric complexes with each polypeptide containing a single bilin (or linear tetrapyrrole) chromophore, which binds autocatalytically via a thioether linkage to a positionally conserved cysteine (1-3). The photosensing portion typically contains Per/Arndt/Sim (PAS) and cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains, which are essential for bilin binding and Pr assembly, and t...

Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag electrode, which are formed by omega-carboxyl alkanethiols of different chain lengths (C(x)). The dynamics of the electron-transfer (ET) reaction of the adsorbed heme protein, initiated by a rapid potential jump to the redox potential, was monitored by time-resolved surface enhanced resonance Raman (SERR) spectroscopy. Under conditions of the present experiments, only the reduced and oxidized forms of the native protein state contribute to the SERR spectra. Thus, the data obtained from the spectra were described by a one-step relaxation process yielding the rate constants of the ET between the adsorbed Cyt-c and the electrode for a driving force of zero electronvolts. For C(16)- and C(11)-SAMs, the respective rate constants of 0.073 and 43 s(-1) correspond to an exponential distance dependence of the ET (beta = 1.28 A(-1)), very similar to that observed for long-range intramolecular ET of redox proteins. Upon further decreasing the chain length, the rate constant only slightly increases to 134 s(-1) at C(6)- and remains essentially unchanged at C(3)- and C(2)-SAMs. The onset of the nonexponential distance dependence is paralleled by a kinetic H/D effect that increases from 1.2 at C(6)- to 4.0 at C(2)-coatings, indicating a coupling of the redox reaction with proton-transfer (PT) steps. These PT processes are attributed to the rearrangement of the hydrogen-bonding network of the protein associated with the transition between the oxidized and reduced state of Cyt-c. Since this unusual kinetic behavior has not been observed for electron-transferring proteins in solution, it is concluded that at the Ag/SAM interface the energy barrier for the PT processes of the adsorbed Cyt-c is raised by the electric field. This effect increases upon reducing the distance to the electrode, until nuclear tunneling becomes the rate-limiting step of the redox process. The electric field dependence of the proton-coupled ET may represent a possible mechanism for controlling biological redox reactions via changes of the transmembrane potential.

The heme protein cytochrome c acts as an electron carrier at the mitochondrial-membrane interface and thus exerts its function under the influence of strong electric fields. To assess possible consequences of electric fields on the redox processes of cytochrome c, the protein can be immobilized to self-assembled monolayers on electrodes and studied by surface-enhanced resonance Raman spectroscopy. Such model systems may mimic some essential features of biological interfaces including local electric field strengths. It is shown that physiologically relevant electric field strengths can effectively modulate the electron-transfer dynamics and induce conformational transitions.

The P r 3 P fr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R C were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in P r and ZZEssa in P fr , with all pyrrole nitrogens being protonated. Photoexcitation of P r is followed by (at least) three thermal relaxation components in the formation of P fr with time constants of 230 s and 3.1 and 260 ms. H 2 O/D 2 O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R C , respectively. Resonance Raman measurements of Meta-R C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P r 3 P fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the P fr formation that switch between the active and the inactive state of the photoreceptor.Phytochromes are photoreceptors that utilize light as a source of information for controlling numerous biological processes (1, 2). The chromophore, a methine-bridged tetrapyrrole ( Fig. 1), acts as a photoswitch between two stable, spectrally distinct forms, denoted as P r and P fr according to the red and far-red absorption maxima, respectively. The P r /P fr interconversion is initiated by the rapid Z/E photoisomerization of the C-D methine bridge (3), followed by chromophore relaxations that are coupled to structural changes of the apoprotein (4). These structural changes are the trigger for signal transduction. Resonance Raman (RR) 2 and IR spectroscopy have provided valuable insight into light-induced chromophore and protein structural changes (e.g. see Refs. 5-10), but molecular and mechanistic details are not yet known and no crystal structure of a phytochrome has been reported so far.While phytochromes were originally thought to be restricted to plants, the discovery of these chromoproteins in cyanobacteria (11) and other bacteria points to the prokaryotic origin of this family of photoreceptors. In contrast to plant phytochromes, typical bacterial phytochromes are light-regulated histidine kinases. Despite the quite different regulatory functions (12, 13), plant and bacterial phytochromes exhibit structural and mechanistic similarities. The phytochromobilin chromophore of plant phytoc...

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK a from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc 3 Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein.

Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on electrodes coated with biocompatible materials to prevent denaturation. However, even when protein denaturation is effectively avoided, heterogeneous protein electron transfer is often coupled to non-Faradaic processes like reorientation, conformational transitions or acid-base equilibria. Disentangling these processes requires methods capable of probing simultaneously the structure and reaction dynamics of the adsorbed species. Here we provide an overview of the recent developments in Raman and infrared surface-enhanced spectroelectrochemical techniques applied to the study of soluble and membrane bound redox heme proteins and enzymes. Possible biological implications of the findings are critically discussed.