Mark R. Ondrias

Evidence for proximal control of ligand specificity in hemeproteins: Absorption and Raman studies of cryogenically trapped photoproducts of ligand bound myoglobins☆

Abstract The absorption and resonance Raman spectra of cryogenically trapped photoproducts of oxy and carboxy derivatives of myoglobin (Mb) are compared and analyzed in an attempt to understand the structural basis for ligand specificity in hemeproteins. Pulsed and cw excitations over a wide temperature range are used in order to differentiate between kinetic hole burning (KHB), optical pumping of structural relaxation, and spontaneous relaxation effects. Using these techniques, we are able to correlate changes in the absorption spectrum (band III at ≈ 760 nm) with low-frequency Raman bands. Based on these correlations we are able to determine which proximal heme pocket parameters are participating in KHB and optical pumping phenomena. Differences in the spectra of the ligand specific photoproducts have revealed differences in the populations of conformational substates (CS) that participate in the geminate recombination (process I) at cryogenic temperatures. A model is presented that relates the ligand specific spectral differences to structural and functional differences in the bound protein. What emerges is evidence that Mb and hemoglobin (Hb) can differentiate between O 2 and CO based on proximal control of the bond forming step between the ligand and the iron.

Mode-selective energy localization during photoexcitation of deoxyhemoglobin and heme model complexes☆

Abstract Vibrational energy redistribution may be important in regulating the function of hemes and related macrocycles. Transient resonance Raman experiments reveal a non-Boltzman vibrational energy distribution among the heme normal modes of photoexcited doexyhemoglobin. Within 10 ns excitation pulses, v 4 exhibits flux-dependent anti-Stokes peak position, Stokes and anti-Stokes linewidths, and anti-Stokes:Stokes ratios corresponding to temperatures as high as 600 K. Other modes, most notably v 7 , do not behave in this manner. Spectra of protoporphyrin IX-2-methyl-imidazole obtained under the same conditions indicate that specific heme-protein interactions cannot be entirely responsible for this behavior.

Characterization of the siroheme active site in spinach nitrite reductase by resonance Raman spectroscopy

The resonance Raman spectra of various species of spinach nitrite reductase (ferredoxin: nitrite oxidoreductase, EC 1.7.7.1) have been obtained with Soret excitation. These spectra allow for the vibrational properties of the unique siroheme chromophore at the enzymes active site. The wholesale reordering of siroheme vibrational properties relative to those of protoporphyrins can be rationalized as resulting from a combination of symmetry lowering and bond order reductions within the siroheme macrocyle.

RESONANCE RAMAN SPECTROSCOPY OF CYTOCHROME BC1 COMPLEXES FROM RHODOSPIRILLUM RUBRUM : INITIAL CHARACTERIZATION AND REDUCTIVE TITRATIONS

Resonance Raman spectra of bc1 complexes from Rhodospirillum rubrum have been obtained. Various resonance conditions and the stoichiometric redox titration of the complex were used to isolate and identify the contributions of the heme c1 and heme b active sites to the observed spectra. The complex was found to partially photoreduce when exposed to laser excitation.

Picosecond optical studies of nickel(II) protoporphyrin IX dimethyl ester

Abstract Time-resolved Raman and adsorption spectroscopies were used to characterize the vibrational and electronic structure of the *B1g excited state of nickel(II) protoporphyrin IX dimethyl ester. Evidence for structural relaxation processes within the initial B1g excited state was observed on picosecond time scales. We conclude that interconversions between structural conformers are important in these processes. Such conformational interconversions in metalloporphyrins may have a major role in the complex mechanisms of many biological systems.

Transient Raman observations of heme vibrational dynamics in five-coordinate iron porphyrins

Abstract Transient resonance Raman spectroscopy has been used to study vibrational dynamics in five-coordinate, high-spin Fe II octaethyl porphyrin with a 2-methyl imidazole axial ligand. Vibrational populations of the porphyrin ground electronic state were probed by examining Stokes and anti-Stokes Raman scattering as a function of incident laser flux using ∼10 nanosecond pulses in resonance with the Soret electronic transition. Within a single pulse, each molecule goes through several excitation-decay cycles, building up a non-equilibrium, excited vibrational energy distribution that is exquisitely sensitive to the vibrational mode lifetimes and to the incident laser flux. A kinetic model illustrates these ideas and provides strong support for the interpretation of the results. The flux dependence of the Raman intensities, positions and linewidths suggests that ν 3 and ν 4 act as “bottleneck” vibrational states, while ν CH and ν 7 couple more effectively to the environment.

Spectroscopic characterization of heme a reconstituted myoglobin

The focus of this study was to examine the functional role of the unusual peripheral substitution of heme A. The effects of heme A stereochemistry on the reconstitution of the porphyrin have been examined in the heme A-apo-myoglobin complex using optical absorption and resonance Raman and electron paramagnetic resonance spectroscopies. The addition of one equivalent of heme A to apo-Mb produces a complex which displays spectroscopic signals consistent with a distribution of high- and low-spin heme chromophores. These results indicate that the incorporation of heme A into apo-Mb significantly perturbs the protein refolding.

Resonance Raman investigation of transient photoinduced ligation changes in nickel octaethyl porphyrin

Abstract The first picosecond and nanosecond time-resolved Raman spectra of photoexcited metalloporphyrins in the coordinating solvent piperidine are presented. Our data demonstrate that time-resolved resonance Raman spectra can be obtained from nickel octaethyl porphyrins and confirm the existence of photoinduced ligation changes in nickel octaethyl porphyrin species on a sub-nanosecond timescale. It is inferred from the power-dependent behavior of various porphyrin vibrational modes that multiple photochemical effects are contributing to the observed transient spectra.

Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1.

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.

Resonance Raman investigation of a soluble cytochrome c552 from alkaliphilic Bacillus firmus RAB.

The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.