We explore heme structure and ligation subsequent to a low-pH conformational transition in sperm whale myoglobin. Below pH 4.0, the iron-histidine bond breaks in metMb and deoxyMb. In MbCO, the majority of the iron-histidine bonds remain intact down to pH 2.6; however, the observation of a weak Fe-CO mode at 526 cm-1 indicates that a small fraction of the sample has the histidine replaced by a weak ligand, possibly water. The existence of a sterically hindered CO subpopulation in MbCO and the continued association of the four-coordinate heme with the protein in deoxyMb suggest that the heme pocket remains at least partially intact in the acid-induced conformation. The global pH-dependent conformational change described here is clearly distinguished from the local "closed" to "open" transition described previously in MbCO [Morikis et al. (1989) Biochemistry 28, 4791-4800]. Further observations of the four-coordinate heme state yield insights on the mechanism of heme photoreduction and the assignment of the 760-nm band in deoxyMb.

For many years, Mössbauer spectroscopy has been applied to measure recoilless absorption of x-ray photons by nuclei. Recently, synchrotron radiation sources have enabled the observation of weaker features separated from the recoilless resonance by the energy of vibrational quanta. This enables a form of vibrational spectroscopy with a unique sensitivity to the probe nucleus. Biological applications are particularly promising, because it is possible to selectively probe vibrations of a single atom at the active site of a complex biomolecule, while avoiding interference from the vibrations of thousands of other atoms. In contrast with traditional site-selective vibrational spectroscopies, nuclear resonance vibrational spectroscopy (NRVS) is not hampered by solvent interference and faces selection rule limitations only if the probe nucleus lies on a symmetry element. Here, we formulate a mathematical language appropriate for understanding NRVS measurements on molecular systems and apply it to analyse NRVS data recorded on ferrous nitrosyl tetraphenylporphyrin, Fe(TPP)(NO). This compound mimics the haem group found at the active site of many proteins involved in the biological usage of oxygen and nitric oxide. Measurements on such model compounds provide a baseline for evaluating the extent to which vibrations are localized at the active site of a protein, with the goal of elucidating the mechanisms of biological processes, such as intersite communication in allosteric proteins.

We use quantitative experimental and theoretical approaches to characterize the vibrational dynamics of the Fe atom in porphyrins designed to model heme protein active sites. Nuclear resonance vibrational spectroscopy (NRVS) yields frequencies, amplitudes, and directions for 57Fe vibrations in a series of ferrous nitrosyl porphyrins, which provide a benchmark for evaluation of quantum chemical vibrational calculations. Detailed normal mode predictions result from DFT calculations on ferrous nitrosyl tetraphenylporphyrin Fe(TPP)(NO), its cation [Fe(TPP)(NO)]+, and ferrous nitrosyl porphine Fe(P)(NO). Differing functionals lead to significant variability in the predicted Fe-NO bond length and frequency for Fe(TPP)(NO). Otherwise, quantitative comparison of calculated and measured Fe dynamics on an absolute scale reveals good overall agreement, suggesting that DFT calculations provide a reliable guide to the character of observed Fe vibrational modes. These include a series of modes involving Fe motion in the plane of the porphyrin, which are rarely identified using infrared and Raman spectroscopies. The NO binding geometry breaks the four-fold symmetry of the Fe environment, and the resulting frequency splittings of the in-plane modes predicted for Fe(TPP)(NO) agree with observations. In contrast to expectations of a simple three-body model, mode energy remains localized on the FeNO fragment for only two modes, an N-O stretch and a mode with mixed Fe-NO stretch and FeNO bend character. Bending of the FeNO unit also contributes to several of the in-plane modes, but no primary FeNO bending mode is identified for Fe(TPP)(NO). Vibrations associated with hindered rotation of the NO and heme doming are predicted at low frequencies, where Fe motion perpendicular to the heme is identified experimentally at 73 and 128 cm-1. Identification of the latter two modes is a crucial first step toward quantifying the reactive energetics of Fe porphyrins and heme proteins.

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Femtosecond laser pulses, resonant with Soret band of the nitric oxide complex of myoglobin (MbNO), were used to probe coherent, low-frequency nuclear motion of the heme group after photolysis. Distinct oscillations with periods of 430 and 150 femtoseconds were observed and are attributed to heme doming and iron-histidine motion, respectively. These results verify that the nuclear motion of the heme is strongly coupled to the ligand binding reaction and demonstrate that such motion is not determined by overdamped (diffusive) dynamics. The relative phases and frequencies of the nuclear motion of the photoproduct suggest that the coherence arises from impulsive electronic forces associated with the spin-state change of the heme iron atom and the depopulation of its dz2 orbital during the bond-breaking event.

We report Mössbauer and EPR measurements on horseradish peroxidase in the native state and the reaction intermediates with peroxide and chlorite. A detailed analysis of the electronic state of the heme iron is given, and comparisons are drawn with related systems. The native enzyme is high-spin ferric and thus has three Kramers doublets. The unusual magnetic properties of the ground doublet and the large energy of the second, (E2-E1)/k approximately equal to 41 K, and third doublet, (E3-E1)/k greater than or equal to 170 K, can be modeled with a quartet admixture of approximately 11% to the spin sextet. All evidence suggests a ferryl, OFeIV, state of the heme iron in compounds I and II and related complexes. The small isomer shift, delta Fe approximately equal to 0.06 mm/s, the (positive) quadrupole splitting, delta EQ approximately equal to 1.4 mm/s, the spin S = 1, and the large positive zero field splitting, D/k approximately equal to 35 K, are all characteristic of the ferryl state. In the green compound I the iron weakly couples to a porphyrin radical with spin S' = 1/2. A phenomenological model with a weak exchange interaction S . J . S', magnitude of less than or equal to 0.1 D, reproduces all Mössbauer and EPR data of compound I, but the structural origin of the exchange and its apparent distribution require further study. Reaction of horseradish peroxidase with chlorite leads to compound X with delta Fe = 0.07 mm/s and delta EQ = 1.53 mm/s, values that are closest to those of compound II. The diamagnetism of compound III and its Mössbauer parameters delta Fe = 0.23 mm/s and delta EQ = -2.31 mm/s at 4.2 K clearly identify it as an oxyheme adduct.

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.

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