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HemAT is a heme-containing oxygen sensor protein that controls aerotaxis. Time-resolved step-scan FTIR studies were performed on the isolated sensor domain and full-length HemAT proteins as well as on the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) mutants to elucidate the effect of the site-specific mutations on the ligand dynamics subsequent to CO photolysis. The mutations aimed to perturb H-bonding and electrostatic interactions near the heme Fe-bound gaseous ligand (CO) and the heme proximal environment. Rebinding of CO to the heme Fe is biphasic in the sensor domain and full-length HemAT as well as in the mutants, with the exception of the Y133F mutant protein. The monophasic rebinding of CO in Y133F suggests that in the absence of the H-bond between Y133 and the heme proximal H123 residue the ligand rebinding process is significantly affected. The role of the proximal environment is also probed by resonance Raman photodissociation experiments, in which the Fe-His mode of the photoproduct of sensor domain HemAT-CO is detected at a frequency higher than that of the deoxy form in the difference resonance Raman spectra. The role of the conformational changes of Y133 (G-helix) and the role of the distal L92 and T95 residues (E-helix) in regulating ligand dynamics in the heme pocket are discussed.

The coordination of nitrite in myoglobin (Mb) has been characterized by resonance Raman spectroscopy and the frequencies of the nitrite bound to the heme Fe as well to the 2-vinyl have been computed by density functional theory (DFT) calculations. The DFT Natural Bond Orbital (NBO) analysis and the extensive isotope-labeling in the resonance Raman experiments indicate that NO 2 (O1NO2) is bound to the heme Fe via O1. Based on the vibrational characterization of the reversible transition between low and high spin FeONO/2-nitrovinyl species, we suggest that the key step that triggers the spin-change is the increase of the proximal FeN - bond length. The frequencies of the O and N sensitive bands of the FeONO/2-nitrovinyl species remained largely unchanged in the low- to high-spin transition. Therefore the "greening" process in the reaction of ferric Mb with NO His93 bond length. The frequencies of the O and N sensitive bands of the FeONO/2-nitrovinyl species remained largely unchanged in the low- to high-spin transition. Therefore the "greening" process in the reaction of ferric Mb with NO 2 - proceeds through the FeONO/2-nitrovinyl species, which can exist in either the high or low-spin state., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The myoglobin (Mb) heme Fe-O-N=O and heme Fe-O-N=O/2-nitrovinyl species have been characterized by resonance Raman spectroscopy. In the heme Fe-O-N=O species, the bound nitrite ligand is removed by solvent exchange, thus reforming metmyoglobin (metMb). The high-spin heme Fe-O-N=O unit is converted into a low-spin heme Fe-O-N=O/2-nitrovinyl species that can be reversibly switched between a low- and a high-spin state without removing the bound nitrite ligand, as observed in the case of the heme Fe-O-N=O species. This spin-state change is likely to be accompanied by a general structural rearrangement in the protein-binding pocket. This example is the first of a globin protein that can reversibly change its metal spin state through an internal perturbation. These findings provide a basis for understanding the structure-function relationship of the spin cross found in other metalloenzymes and Fe(III) -porphyrin complexes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Members of the cytochrome c oxidase family exhibit nitrite reductase activity. In this work, we have characterized a ferrous heme a3-nitro species in ba3-oxidase by resonance Raman spectroscopy. This provides the first evidence for the structure of a nitrite-bound species in the binuclear heme/copper center of cytochrome c oxidases.

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O2 activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O2 with the mixed valence form (CuA(2+), heme a(3+), heme a3(2+)-CuB(1+)) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm(-1) at the peroxy oxidation level (PM). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the PM intermediate is also formed in the reaction of Y167F with O2. These results suggest that in the fully reduced enzyme, the proton pumping ν(Fe(IV)=O) = 804 cm(-1) to ν(Fe(IV)=O) = 790 cm(-1) transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a3 but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a3. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a3-Asp-399-H2O site and, thus, to the exit/output proton channel (H2O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a3 controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.

FTIR and light-minus-dark FTIR spectroscopy have been employed to investigate the reaction of oxidized and fully reduced ba(3) oxidase with cyanide. The characterization of the structures of the bound CN(-) in the binuclear heme Fe-Cu(B) center is essential, given that a central issue in the function of ba(3) oxidase is the extent to which the partially reduced substrates interact with the two metals. In the reaction of oxidized ba(3) oxidase with cyanide the initially formed heme a(3)(3+)-C≡N-Cu(B)(2+) species with ν(CN) frequency at 2152 cm(-1) was replaced by a photolabile complex with a frequency at 2075 cm(-1) characteristic of heme a(3)(2+)-CN(-). Photolysis of the heme a(3)(2+)-CN(-) adduct produced a band at 2146 cm(-1) attributed to the formation of a transient Cu(B)(2+)-CN(-) complex. All forms are pH independent between pH 5.5-9.5 and at pD 7.5 indicating the absence of ionizable groups that influence the properties of the cyanide complexes. In contrast to previous reports, our results show that CN(-) does not bind simultaneously to both heme a(3)(2+) and Cu(B)(2+) to form the mixed valence a(3)(2+)-CN·Cu(B)(2+)CN species. The photolysis products of the heme a(3)(2+)-CN(-)/Cu(B)(2+) and heme a(3)(2+)-CN(-)/Cu(B)(1+) species are different suggesting that relaxation dynamics in the binuclear center following ligand photodissociation are dependent on the oxidation state of Cu(B).

Fourier transform infrared (FTIR) spectra, "light" minus "dark" difference FTIR spectra, and time-resolved step-scan (TRS(2)) FTIR spectra are reported for carbonmonoxy aldoxime dehydratase. Two C-O modes of heme at 1945 and 1964 cm(-1) have been identified and remained unchanged in H(2)O/D(2)O exchange and in the pH 5.6-8.5 range, suggesting the presence of two conformations at the active site. The observed C-O frequencies are 5 and 16 cm(-1) lower and higher, respectively, than that obtained previously (Oinuma, K.-I.; et al. FEBS Lett.2004, 568, 44-48). We suggest that the strength of the Fe-His bond and the neutralization of the negatively charged propionate groups modulate the ν(Fe-CO)/ν(CO) back-bonding correlation. The "light" minus "dark" difference FTIR spectra indicate that the heme propionates are in both the protonated and deprotonated forms, and the photolyzed CO becomes trapped within a ligand docking site (ν(CO) = 2138 cm(-1)). The TRS(2)-FTIR spectra show that the rate of recombination of CO to the heme is k(1945 cm(-1)) = 126 ± 20 s(-1) and k(1964 cm(-1)) = 122 ± 20 s(-1) at pH 5.6, and k(1945 cm(-1)) = 148 ± 30 s(-1) and k(1964 cm(-1)) = 158 ± 32 s(-1) at pH 8.5. The rate of decay of the heme propionate vibrations is on a time scale coincident with the rate of rebinding, suggesting that there is a coupling between ligation dynamics in the distal heme environment and the environment sensed by the heme propionates. The implications of these results with respect to the proximal His-Fe heme environment including the propionates and the positively charged or proton-donating residues in the distal pocket which are crucial for the synthesis of nitriles are discussed.

Resonance Raman (RR) and "light" minus "dark" Fourier transform infrared (FTIR) difference spectra are reported for the CO-bound caa(3) oxidase from Thermus thermophilus. Two Fe-CO stretching modes at 518 and 507 cm(-1), the Fe-C-O bending mode at 570 cm(-1), and three C-O modes of heme a(3) at 1958, 1967, and 1973 cm(-1) have been identified in the RR and FTIR spectra, respectively. The FTIR "light" minus "dark" spectrum indicates the formation of Cu(B)CO as revealed by its ν(CO) at 2060/2065 cm(-1). We assign the bands at 518 (ν(Fe-CO)) and 1967/1973 cm(-1) (ν(C-O)) as the α-conformation. We also assign the bands at 507 and 1958 cm(-1) (ν(C-O)) as originating from the β-conformation of the enzyme. A frequency upshift of the heme a(3) Fe-His mode is observed subsequent to CO photolysis from 209 cm(-1) in the equilibrium deoxy enzyme to 214 cm(-1) in the photoproduct. The caa(3) data, distinctly different from those of ba(3) oxidase, are discussed in terms of the coupling of the α- and β-conformations that occur in heme-copper oxidases with catalytic function. The dynamics between the heme a(3) and heme a propionates as revealed by the perturbation of the bending vibrations δ(prop) of hemes a and a(3) at 385 and 392 cm(-1), respectively, induced upon CO binding to heme a(3) is discussed in terms of the protonic connectivity between the heme a ring-D propionate/Arg site with that of the heme a(3) ring-D propionate-H(2)O site that leads to the highly conserved in the heme-copper oxidases water pool., (© 2011 American Chemical Society)