FT-IR Characterization of the Light-Induced Ni-L2 and Ni-L3 States of [NiFe] Hydrogenase from Desulfovibrio vulgaris Miyazaki F

Different light-induced Ni-L states of [NiFe] hydrogenase from its Ni-C state have previously been observed by EPR spectroscopy. Herein, we succeeded in detecting simultaneously two Ni-L states of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by FT-IR spectroscopy. A new light-induced ν_CO band at 1890 cm^–1 and ν_CN bands at 2034 and 2047 cm^–1 were detected in the FT-IR spectra of the H₂-activated enzyme under N₂ atmosphere at basic conditions, in addition to the 1910 cm^–1 ν_CO band and 2047 and 2061 cm^–1 ν_CN bands of the Ni-L2 state. The new bands were attributed to the Ni-L3 state by comparison of the FT-IR and EPR spectra. The ν_CO and ν_CN frequencies of the Ni-L3 state are the lowest frequencies observed among the corresponding frequencies of standard-type [NiFe] hydrogenases in various redox states. These results indicate that a residue, presumably Ni-coordinating Cys546, is protonated and deprotonated in the Ni-L2 and Ni-L3 states, respectively. Relatively small Δ<i>H</i> (6.4 ± 0.8 kJ mol^–1) and Δ<i>S</i> (25.5 ± 10.3 J mol^–1 K^–1) values were obtained for the conversion from the Ni-L2 to Ni-L3 state, which was in agreement with the previous proposals that deprotonation of Cys546 is important for the catalytic reaction of the enzyme

Interaction between the Heme and a G‑Quartet in a Heme–DNA Complex

The structure of a complex between heme­(Fe³⁺) and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d­(TTAGGG), has been characterized by ¹H NMR. The study demonstrated that the heme­(Fe³⁺) is sandwiched between the 3′-terminal G-quartets of the G-quadruplex DNA. Hence, the net +1 charge of the heme­(Fe³⁺) in the complex is surrounded by the eight carbonyl oxygen atoms of the G-quartets. Interaction between the heme Fe³⁺ and G-quartets in the complex was clearly manifested in the solvent ¹H/²H isotope effect on the NMR parameters of paramagnetically shifted heme methyl proton signals, and interaction of the heme Fe³⁺ with the eight carbonyl oxygen atoms of the two G-quartets was shown to provide a strong and axially symmetric ligand field surrounding the heme Fe³⁺, yielding a heme­(Fe³⁺) low-spin species with a highly symmetric heme electronic structure. This finding provides new insights as to the design of the molecular architecture and functional properties of various heme–DNA complexes

Inversion of the Stereochemistry around the Sulfur Atom of the Axial Methionine Side Chain through Alteration of Amino Acid Side Chain Packing in Hydrogenobacter thermophilus Cytochrome <i>c</i>₅₅₂ and Its Functional Consequences

In cytochrome <i>c</i>, the coordination of the axial Met S_δ atom to the heme Fe atom occurs in one of two distinctly different stereochemical manners, i.e., <i>R</i> and <i>S</i> configurations, depending upon which of the two lone pairs of the S_δ atom is involved in the bond; hence, the Fe-coordinated S_δ atom becomes a chiral center. In this study, we demonstrated that an alteration of amino acid side chain packing induced by the mutation of a single amino acid residue, i.e., the A73V mutation, in Hydrogenobacter thermophilus cytochrome <i>c</i>₅₅₂ (HT) forces the inversion of the stereochemistry around the S_δ atom from the <i>R</i> configuration [Travaglini-Allocatelli, C., et al. (2005) <i>J. Biol. Chem</i>. <i>280</i>, 25729–25734] to the <i>S</i> configuration. Functional comparison between the wild-type HT and the A73V mutant possessing the <i>R</i> and <i>S</i> configurations as to the stereochemistry around the S_δ atom, respectively, demonstrated that the redox potential (<i>E</i>_m) of the mutant at pH 6.00 and 25 °C exhibited a positive shift of ∼20 mV relative to that of the wild-type HT, i.e., 245 mV, in an entropic manner. Because these two proteins have similar enthalpically stabilizing interactions, the difference in the entropic contribution to the <i>E</i>_m value between them is likely to be due to the effect of the conformational alteration of the axial Met side chain associated with the inversion of the stereochemistry around the S_δ atom due to the effect of mutation on the internal mobility of the loop bearing the axial Met. Thus, the present study demonstrated that the internal mobility of the loop bearing the axial Met, relevant to entropic control of the redox function of the protein, is affected quite sensitively by the contextual stereochemical packing of amino acid side chains in the proximity of the axial Met

Characterization of Heme–DNA Complexes Composed of Some Chemically Modified Hemes and Parallel G‑Quadruplex DNAs

Heme {Fe­(II)- or Fe­(III)-protoporphyrin IX complex [heme­(Fe²⁺) or heme­(Fe³⁺), respectively]} binds selectively to the 3′-terminal G-quartet of a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d­(TTAGGG), through a π–π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The binding affinities of some chemically modified hemes­(Fe³⁺) for DNA and the structures of complexes between the modified hemes­(Fe²⁺) and DNA, with carbon monoxide (CO) coordinated to the heme Fe atom on the side of the heme opposite the G6 G-quartet, have been characterized to elucidate the interaction between the heme and G-quartet in the complexes through analysis of the effects of the heme modification on the structural properties of the complex. The study revealed that the binding affinities and structures of the complexes were barely affected by the heme modification performed in the study. Such plasticity in the binding of heme to the G-quartet is useful for the versatile design of the complex through heme chemical modification and DNA sequence alteration. Furthermore, exchangeable proton signals exhibiting two-proton intensity were observed at approximately −3.5 ppm in the ¹H nuclear magnetic resonance (NMR) spectra of the CO adducts of the complexes. Through analysis of the NMR results, together with theoretical consideration, we concluded that the heme­(Fe²⁺) axial ligand <i>trans</i> to CO in the complex is a water molecule (H₂O). Identification of the Fe-bound H₂O accommodated between the heme and G-quartet planes in the complex provides new insights into the structure–function relationship of the complex

Electronic Control of Ligand-Binding Preference of a Myoglobin Mutant

The L29F mutant of sperm whale myoglobin (Mb), where the leucine 29 residue was replaced by phenylalanine (Phe), was shown to exhibit remarkably high affinity to oxygen (O₂), possibly due to stabilization of the heme Fe atom-bound O₂ in the mutant protein through a proposed unique electrostatic interaction with the introduced Phe29, in addition to well-known hydrogen bonding with His64 [Carver, T. E.; Brantley, R. E.; Singleton, E. W.; Arduini, R. M.; Quillin, M. L.; Phillips, G. N., Jr.; Olson, J. S. <i>J. Biol. Chem.</i>, <b>1992</b>, 267, 14443–14450]. We analyzed the O₂ and carbon monoxide (CO) binding properties of the L29F mutant protein reconstituted with chemically modified heme cofactors possessing a heme Fe atom with various electron densities, to determine the effect of a change in the electron density of the heme Fe atom (ρ_Fe) on the O₂ versus CO discrimination. The study demonstrated that the preferential binding of O₂ over CO by the protein was achieved through increasing ρ_Fe, and the ordinary ligand-binding preference, that is, the preferential binding of CO over O₂, by the protein was achieved through decreasing ρ_Fe. Thus, the O₂ and CO binding preferences of the L29F mutant protein could be controlled through electronic modulation of intrinsic heme Fe reactivity through a change in ρ_Fe. The present study highlighted the significance of the tuning of the intrinsic heme Fe reactivity through the heme electronic structure in functional regulation of Mb

Relationship between Oxygen Affinity and Autoxidation of Myoglobin

Studies using myoglobins reconstituted with a variety of chemically modified heme cofactors revealed that the oxygen affinity and autoxidation reaction rate of the proteins are highly correlated to each other, both decreasing with decreasing the electron density of the heme iron atom. An Fe³⁺–O₂^–-like species has been expected for the Fe²⁺–O₂ bond in the protein, and the electron density of the heme iron atom influences the resonance process between the two forms. A shift of the resonance toward the Fe²⁺–O₂ form results in lowering of the O₂ affinity due to an increase in the O₂ dissociation rate. On the other hand, a shift of the resonance toward the Fe³⁺–O₂^–-like species results in acceleration of the autoxidation through increasing H⁺ affinity of the bound ligand

Activation Mechanism of the <i>Streptomyces</i> Tyrosinase Assisted by the Caddie Protein

Tyrosinase (EC 1.14.18.1), which possesses two copper ions at the active center, catalyzes a rate-limiting reaction of melanogenesis, that is, the conversion of a phenol to the corresponding <i>ortho</i>-quinone. The enzyme from the genus <i>Streptomyces</i> is generated as a complex with a “caddie” protein that assists the transport of two copper ions into the active center. In this complex, the Tyr⁹⁸ residue in the caddie protein was found to be accommodated in the pocket of the active center of tyrosinase, probably in a manner similar to that of l-tyrosine as a genuine substrate of tyrosinase. Under physiological conditions, the addition of the copper ion to the complex releases tyrosinase from the complex, in accordance with the aggregation of the caddie protein. The release of the copper-bound tyrosinase was found to be accelerated by adding reducing agents under aerobic conditions. Mass spectroscopic analysis indicated that the Tyr⁹⁸ residue was converted to a reactive quinone, and resonance Raman spectroscopic analysis indicated that the conversion occurred through the formations of μ-η²:η²-peroxo-dicopper­(II) and Cu­(II)-semiquinone. Electron paramagnetic resonance analysis under anaerobic conditions and Fourier transform infrared spectroscopic analysis using CO as a structural probe under anaerobic conditions indicated that the copper transportation process to the active center is a reversible event in the tyrosinase/caddie complex. Aggregation of the caddie protein, which is triggered by the conversion of the Tyr⁹⁸ residue to dopaquinone, may ensure the generation of fully activated tyrosinase

Relationship between the Electron Density of the Heme Fe Atom and the Vibrational Frequencies of the Fe-Bound Carbon Monoxide in Myoglobin

We analyzed the vibrational frequencies of the Fe-bound carbon monoxide (CO) of myoglobin reconstituted with a series of chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities. The study revealed that the stretching frequency of Fe-bound CO (ν_CO) increases with decreasing electron density of the heme Fe atom (ρ_Fe). This finding demonstrated that the ν_CO value can be used as a sensitive measure of the ρ_Fe value and that the π back-donation of the heme Fe atom to CO is affected by the heme π-system perturbation induced through peripheral side chain modifications

Electronic Control of Discrimination between O₂ and CO in Myoglobin Lacking the Distal Histidine Residue

We analyzed the oxygen (O₂) and carbon monoxide (CO) binding properties of the H64L mutant of myoglobin reconstituted with chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities, in order to elucidate the effect of the removal of the distal His64 on the control of both the O₂ affinity and discrimination between O₂ and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (ρ_Fe). The study revealed that, as in the case of the native protein, the O₂ affinity of the H64L mutant protein is regulated by the ρ_Fe value in such a manner that the O₂ affinity of the protein decreases, due to an increase in the O₂ dissociation rate constant, with a decrease in the ρ_Fe value, and that the O₂ affinities of the mutant and native proteins are affected comparably by a given change in the ρ_Fe value. On the other hand, the CO affinity of the H64L mutant protein was found to increase, due to a decrease in the CO dissociation rate constant, with a decrease in the ρ_Fe value, whereas that of the native protein was essentially independent of a change in the ρ_Fe value. As a result, the regulation of the O₂/CO discrimination in the protein through the ρ_Fe value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the ρ_Fe value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does