Proton translocation in cytochrome c oxidase: insights from proton exchange kinetics and vibrational spectroscopy.

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain. It reduces oxygen to water and harnesses the released energy to translocate protons across the inner mitochondrial membrane. The mechanism by which the oxygen chemistry is coupled to proton translocation is not yet resolved owing to the difficulty of monitoring dynamic proton transfer events. Here we summarize several postulated mechanisms for proton translocation, which have been supported by a variety of vibrational spectroscopic studies. We recently proposed a proton translocation model involving proton accessibility to the regions near the propionate groups of the heme a and heme a3 redox centers of the enzyme based by hydrogen/deuterium (H/D) exchange Raman scattering studies (Egawa et al., PLoS ONE 2013). To advance our understanding of this model and to refine the proton accessibility to the hemes, the H/D exchange dependence of the heme propionate group vibrational modes on temperature and pH was measured. The H/D exchange detected at the propionate groups of heme a3 takes place within a few seconds under all conditions. In contrast, that detected at the heme a propionates occurs in the oxidized but not the reduced enzyme and the H/D exchange is pH-dependent with a pKa of ~8.0 (faster at high pH). Analysis of the thermodynamic parameters revealed that, as the pH is varied, entropy/enthalpy compensation held the free energy of activation in a narrow range. The redox dependence of the possible proton pathways to the heme groups is discussed. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation.

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: L-arginine (L-Arg) → N-hydroxy-L-arginine → citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the L-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498-33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the L-Arg reaction but also during the N-hydroxy-L-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS (Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L., and Yeh, S. R. (2007) J. Am. Chem. Soc. 129, 6943-6951).

A Case of an Extremely Preterm Infant with Intussusception Triggered by Acquired Cytomegalovirus Infection.

Intussusception is a common cause of intestinal obstruction in infants aged 6-18 months. However, intussusception in preterm neonates (IPN) is an exceedingly rare disorder. The etiology of IPN remains unclear, but common prenatal injuries, such as those causing intestinal hypoxia/hypoperfusion, dysmotility, and strictures, have been proposed as possible contributing factors. Diagnosis is often delayed because the symptoms closely resemble those of necrotizing enterocolitis (NEC). Given the divergent treatments for IPN and NEC, establishing an early and accurate diagnosis is crucial. IPN is predominantly located in the small intestine (91.6%), and ultrasonography proves useful in its diagnosis. We present a case of a very preterm infant who developed intussusception triggered by acquired cytomegalovirus (aCMV) infection, necessitating surgical treatment. The cause of intussusception in this case was diagnosed as aCMV enteritis because no organic lesions were observed in the advanced part of the intussusception. The presence of CMV was confirmed by CMV-DNA-PCR examination of the resected intestinal tract. Intestinal edema and decreased intestinal peristalsis due to aCMV enteritis are likely the primary causes of the intussusception.

The rate-limiting step in O(2) reduction by cytochrome ba(3) from Thermus thermophilus.

Cytochrome ba(3) (ba(3)) of Thermus thermophilus (T. thermophilus) is a member of the heme-copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a(3)) and a copper (Cu(B)). The heme-copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba(3) with O(2) using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~1ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k=400s(-1)) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O(2) binds rapidly to heme a(3) in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O(2) binds transiently to a trap, likely Cu(B), prior to its migration to heme a(3) for the oxidative reaction, highlighting the critical role of Cu(B) in regulating the oxygen reaction kinetics in the oxidase superfamily.

Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli.

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3). Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO(aa3) to ~0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a(3)-Cu(B) active site. In contrast, the E286C mutation of EcQO(bo3) exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO(aa3), the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO(bo3) is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.

Radical formation in cytochrome c oxidase.

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O(2) redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O(2) with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50µs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO(2)(-) ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O(2) with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H(2)O(2)) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50µs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O(2) and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a(3). This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".

Two tyrosyl radicals stabilize high oxidation states in cytochrome C oxidase for efficient energy conservation and proton translocation.

The reaction of oxidized bovine cytochrome c oxidase (bCcO) with hydrogen peroxide (H(2)O(2)) was studied by electron paramagnetic resonance (EPR) to determine the properties of radical intermediates. Two distinct radicals with widths of 12 and 46 G are directly observed by X-band EPR in the reaction of bCcO with H(2)O(2) at pH 6 and pH 8. High-frequency EPR (D-band) provides assignments to tyrosine for both radicals based on well-resolved g-tensors. The wide radical (46 G) exhibits g-values similar to a radical generated on L-Tyr by UV-irradiation and to tyrosyl radicals identified in many other enzyme systems. In contrast, the g-values of the narrow radical (12 G) deviate from L-Tyr in a trend akin to the radicals on tyrosines with substitutions at the ortho position. X-band EPR demonstrates that the two tyrosyl radicals differ in the orientation of their ß-methylene protons. The 12 G wide radical has minimal hyperfine structure and can be fit using parameters unique to the post-translationally modified Y244 in bCcO. The 46 G wide radical has extensive hyperfine structure and can be fit with parameters consistent with Y129. The results are supported by mixed quantum mechanics and molecular mechanics calculations. In addition to providing spectroscopic evidence of a radical formed on the post-translationally modified tyrosine in CcO, this study resolves the much debated controversy of whether the wide radical seen at low pH in the bovine enzyme is a tyrosine or tryptophan. The possible role of radical formation and migration in proton translocation is discussed.

Evidence for a ferryl intermediate in a heme-based dioxygenase.

In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions.

The single-domain globin from the pathogenic bacterium Campylobacter jejuni: novel D-helix conformation, proximal hydrogen bonding that influences ligand binding, and peroxidase-like redox properties.

The food-borne pathogen Campylobacter jejuni possesses a single-domain globin (Cgb) whose role in detoxifying nitric oxide has been unequivocally demonstrated through genetic and molecular approaches. The x-ray structure of cyanide-bound Cgb has been solved to a resolution of 1.35 A. The overall fold is a classic three-on-three alpha-helical globin fold, similar to that of myoglobin and Vgb from Vitreoscilla stercoraria. However, the D region (defined according to the standard globin fold nomenclature) of Cgb adopts a highly ordered alpha-helical conformation unlike any previously characterized members of this globin family, and the GlnE7 residue has an unexpected role in modulating the interaction between the ligand and the TyrB10 residue. The proximal hydrogen bonding network in Cgb demonstrates that the heme cofactor is ligated by an imidazolate, a characteristic of peroxidase-like proteins. Mutation of either proximal hydrogen-bonding residue (GluH23 or TyrG5) results in the loss of the high frequency nu(Fe-His) stretching mode (251 cm(-1)), indicating that both residues are important for maintaining the anionic character of the proximal histidine ligand. Cyanide binding kinetics for these proximal mutants demonstrate for the first time that proximal hydrogen bonding in globins can modulate ligand binding kinetics at the distal site. A low redox midpoint for the ferrous/ferric couple (-134 mV versus normal hydrogen electrode at pH 7) is consistent with the peroxidase-like character of the Cgb active site. These data provide a new insight into the mechanism via which Campylobacter may survive host-derived nitrosative stress.

Novel methodology to control the adsorption structure of cationic porphyrins on the clay surface using the "size-matching rule".

Saponite-type clays that have different cation exchange capacities were successfully synthesized by hydrothermal synthesis. The structure and properties were analyzed by X-ray diffraction, X-ray fluorescence, (27)Al NMR, FT-IR, thermogravimetric and differential thermal analysis, atomic force microscopy, and cation exchange capacity measurement. The intercharge distances on the synthetic saponite (SS) surfaces were calculated to be 0.8-1.9 nm on the basis of a hexagonal array. The complex formation behavior between SS and cationic porphyrins was examined. It turns out that the average intermolecular distance between porphyrin molecules on the SS surface can be controlled, depending on the charge density of the SS. In the case of tetrakis(1-methylpyridinium-4-yl)porphyrin (H(2)TMPyP(4+)), the average intermolecular distances on the SS surface can be controlled from 2.3 to 3.0 nm on the basis of a hexagonal array. It was also found that absorption maxima of porphyrins depend on the charge density of the SS. The adsorption behavior of porphyrin on the SS surface can be rationally understood by the previously reported "size-matching rule". This methodology using host-guest interaction can realize a unique adsorption structure control of the porphyrin molecule on the SS surface, where the gap distance between guest porphyrin molecules is rather large. These findings will be highly valuable to construct photochemical reaction systems such as energy transfer in the complexes.

Critical structural role of R481 in cytochrome c oxidase from Rhodobacter sphaeroides.

The R481 residue in cytochrome c oxidase from Rhodobacter sphaeroides forms hydrogen bonds with the propionate groups of both heme a and heme a(3). It has been postulated that R481 is the proton loading site in the proton exit pathway essential for proton translocation. A recent functional study showed that the mutations of R481 to His, Leu and Gln cause the reduction of the activity to approximately 5-18% of the native level, and the absence of proton pumping in R481Q but retention of approximately 40% efficiency in R481H and R481L (H.J. Lee, L. Ojemyr, A. Vakkasoglu, P. Brzezinski and R. B. Gennis, manuscript submitted). To decipher the molecular mechanism underlying the perturbed functionalities, we have used resonance Raman spectroscopy to examine the structural properties of the three mutants. The data show that the frequencies of the formyl CO stretching modes of both the heme a and a(3) in the mutants are characteristic of formyl groups exposed to an aqueous environment, indicating that the mutations disrupt the native H-bonding interaction between the formyl group of heme a and R52, as well as the hydrophobic environment surrounding the formyl group of heme a(3). In addition to the change in the environments of heme a and a(3), the Raman data show that the mutations induce a partial conversion of the heme a(3) from a high-spin to a low-spin state, suggesting that the mutations are associated with the rearrangement of the Cu(B)-heme a(3) binuclear center. The Raman results reported here demonstrate that R481 plays a critical role in supporting efficient proton pumping, by holding the heme groups in a proper environment.

Communication between R481 and Cu(B) in cytochrome bo(3) ubiquinol oxidase from Escherichia coli.

The R481 residue of cytochrome bo(3) ubiquinol oxidase from E. coli is highly conserved in the heme-copper oxidase superfamily. It has been postulated to serve as part of a proton loading site that regulates proton translocation across the protein matrix of the enzyme. Along these lines, proton pumping efficiency has been demonstrated to be abolished in many R481 mutants. However, R481Q in bo(3) from E. coli has been shown to be fully functional, implying that the positive charge of the arginine is not required for proton translocation [ Puustinen , A. and Wikstrom , M. ( 1999 ) Proc. Natl. Acad. Sci. U.S.A. 96 , 35 - 37 ]. In an effort to delineate the structural role of R481 in the bo(3) oxidase, we used resonance Raman spectroscopy to compare the nonfunctional R481L mutant and the functional R481Q mutant, to the wild type protein. Resonance Raman data of the oxidized and reduced forms of the R481L mutant indicate that the mutation introduces changes to the heme o(3) coordination state, reflecting a change in position and/or coordination of the Cu(B) located on the distal side of heme o(3), although it is approximately 10 A away from R481. In the reduced-CO adduct of R481L, the frequencies of the Fe-CO and C-O stretching modes indicate that, unlike the wild type protein, the Cu(B) is no longer close to the heme-bound CO. In contrast, resonance Raman data obtained from the various oxidation and ligation states of the R481Q mutant are similar to those of the wild type protein, except that the mutation causes an enhancement of the relative intensity of the beta conformer of the CO-adduct, indicating a shift in the equilibrium between the alpha and beta conformers. The current findings, together with crystallographic structural data of heme-copper oxidases, indicate that R481 plays a keystone role in stabilizing the functional structure of the Cu(B) site through a hydrogen bonding network involving ordered water molecules. The implications of these data on the proton translocation mechanism are considered.

Design and evaluation of a passive alcove-based microfluidic mixer.

A novel passive microfluidic silicon mixer has been designed, optimized and fabricated. The architecture of the mixer consists of a simple "T" junction, made up by a 20 microm wide by 82 microm deep channel, followed by three repeats of an alcove, each with a triangular obstruction, arranged in a zigzag fashion. Numerical simulations were employed to optimize the geometry, particularly the dimensions of the alcoves, the relative orientation and the spacing between them, and the degree of intrusion associated with them. The simulation results demonstrate that chaotic flow due to recirculation within the alcoves results in transverse velocity that promotes effective fluid mixing. The microfluidic mixer with the simulation-optimized geometry was fabricated with photolithographic techniques and characterized by optical imaging, fluorescence, and Raman microscope spectroscopy. At a sample flow rate of 20 microL/s, the mixer exhibits a short mixing deadtime of approximately 22 micros and a high mixing efficiency under both low and high viscosity conditions. The alcove-based microfluidic silicon mixer offers unique advantages for its short deadtime and slow sample consumption rate. In addition, it provides a valuable component for laboratory-on-a-chip applications for its ease of development into multiple networks for massively parallel analytical processes.

Identification of heme propionate vibrational modes in the resonance Raman spectra of cytochrome c oxidase.

The propionate groups of heme a and a(3) in cytochrome c oxidase (CcO) have been postulated to mediate both the electron and proton transfer within the enzyme. To establish structural markers for the propionate groups, their associated vibrational modes were identified in the resonance Raman spectra of CcO from bovine (bCcO) and Rhodobacter sphaeroides (RsCcO). The distinction between the modes from the propionates of heme a and heme a(3), as well as those from the propionates on the pyrrole rings A and D in each heme, was made on the basis of H2O-D2O isotope substitution experiments combined with wavelength-selective resonance enhancement (for bCcO) or mutagenesis studies (for RsCcO).

General mathematical formula for near equilibrium relaxation kinetics of basic enzyme reactions and its applications to find conformational selection steps.

A general mathematical formula of basic enzyme reactions was derived with nearly no dependence on conditions nor assumptions on relaxation kinetic processes near equilibrium in a simple single-substrate-single-product enzyme reaction. The new formula gives precise relationships between the rate constants of the elementary reaction steps and the apparent relaxation rate constant, rather than the initial velocity that is generally used to determine enzymatic parameters according to the Michaelis-Menten theory. The present formula is shown to be complementary to the Michaelis-Menten formulae in a sense that the initial velocity and the relaxation rate constant data together could determine the enzyme-substrate dissociation constant KES, which has been usually conditionally approximated by the Michaelis constant KM within the framework of the Michaelis-Menten formulae. We also describe relaxation kinetics of enzyme reactions that include the conformational selection processes, in which only one enzymatic conformer among a conformational ensemble can bind with either the substrate or product. The present mathematical approaches, together with numerical computation analyses, suggested that the presence of conformational selection steps in enzymatic reactions can be experimentally detected simply by enzymatic assays with catalytic amounts of enzyme.

Effect of Protein Isotope Labeling on the Catalytic Mechanism of Lactate Dehydrogenase.

We investigate how isotopic labeling of the enzyme lactate dehydrogenase (LDH) affects its function. LDH is of special interest because there exists a line of residues spanning the protein that are involved in the transition state (TS) of the chemical reaction coordinate (so-called promoting vibration). Hence, studies have been carried out on this protein (as well as others) using labeled protein (so-called heavy protein) along with measurements of single turnover kcat yielding a KIE (=kcatlight/kcatheavy) aimed at understanding the effect of labeling generally and more specifically this line of residues. Here, it is shown that 13C, 15N, and 2H atom labeling of hhLDH (human heart) affects its internal structure which in turn affects its dynamics and catalytic mechanism. Spectral studies employing advanced FTIR difference spectroscopy show that the height of the electronic potential surface of the TS is lowered (probably by ground state destabilization) by labeling. Moreover, laser-induced T-jump relaxation kinetic spectroscopy shows that the microsecond to millisecond nuclear motions internal to the protein are affected by labeling. While the effects are small, they are sufficient to contribute to the observed KIE values as well or even more than promoting vibration effects.

Hemoglobins from Mycobacterium tuberculosis and Campylobacter jejuni: a comparative study with resonance Raman spectroscopy.

Three groups of hemoglobins (Hbs) have been identified in unicellular organisms: (1) the truncated Hbs (trHb) that display a novel two-over-two alpha-helical structure, (2) the flavohemoglobins (FHb) that comprise a Hb domain with a classical three-over-three alpha-helical structure and a covalently attached flavin-containing reductase domain, and (3) the single-domain Hbs (sdHb) that exhibit high sequence homology and structural similarity to the Hb domain of FHb. On the basis of phylogenetic analysis, the trHbs can be further divided into three subgroups: TrHb-I, TrHb-II, and TrHb-III. The various classes of Hbs may coexist in the same organism, suggesting distinct functions for each class of Hb. This chapter reviews the structural and functional properties of a TrHb-I (trHbN) and a TrHb-II (trHbO) from Mycobacterium tuberculosis, as well as a TrHb-III (trCtb) and a sdHb (Cgb) from Campylobacter jejuni on the basis of resonance Raman spectroscopic studies.

A Fatal Case of Congenital Langerhans Cell Histiocytosis with Disseminated Cutaneous Lesions in a Premature Neonate.

Background. The outcome of neonates with congenital cutaneous Langerhans cell histiocytosis (LCH) is variable. Observations. We report a case of LCH in a female premature neonate born at 33-week gestation. She had disseminated cutaneous lesions, which consisted of hemorrhagic papules and vesicles, with sparse healthy skin areas, and the hands and feet were contracted with scarring and blackened. She was in respiratory failure although no apparent pulmonary or bone lesions on X-rays were noted. Skin biopsy confirmed a diagnosis of LCH due to observation of CD1a+ Langerhans cells, which lacked expression of E-cadherin and CD56. The patient died 57 hours after birth. Conclusions. Based on this case and the literature survey, the outcome of premature babies with congenital cutaneous LCH lesions is noted to be unfavorable, with the majority of such cases suffering from multisystem disease.

Structural and functional properties of hemoglobins from unicellular organisms as revealed by resonance Raman spectroscopy.

Hemoglobins have been discovered in organisms from virtually all kingdoms. Their presence in unicellular organisms suggests that the gene for hemoglobin is very ancient and that the hemoglobins must have functions other than oxygen transport, in view of the fact that O2 delivery is a diffusion-controlled process in these organisms. Based on sequence alignment, three groups of hemoglobins have been characterized in unicellular organisms. The group-one hemoglobins, termed truncated hemoglobins, consist of proteins with 110-140 amino acid residues and a novel two-over-two alpha-helical sandwich motif. The group-two hemoglobins, termed flavohemoglobins, consist of a hemoglobin domain, with a classical three-over-three alpha-helical sandwich motif, and a flavin-containing reductase domain that is covalently attached to it. The group-three hemoglobins consist of myoglobin-like proteins that have high sequence homology and structural similarity to the hemoglobin domain of flavohemoglobins. In this review, recent resonance Raman studies of each group of these proteins are presented. Their implications are discussed in the context of the structural and functional properties of these novel hemoglobins.