Ferric Heme Iron Research Articles

Initial characterization of the ferric H175G cytochrome c peroxidase cavity mutant using magnetic circular dichroism spectroscopy: phosphate from the buffer as an axial ligand

Electronic absorption and magnetic circular dichroism (MCD) data are reported for the H175G cavity mutant of yeast cytochrome c peroxidase (H175G CCP) in its ligand-free ferric state at 4 °C under slightly acidic conditions (pH 5.9). Initial analysis of the MCD spectrum of this state suggested a five-coordinate structure with ligation by an aspartate or glutamate. However, examination of the crystal structure of wild-type CCP revealed no plausible carboxylate-containing amino-acid residues close enough to the heme iron to serve as the ligand. An alternative interpretation of the MCD data is that the phosphate ion from the buffer is bound to the ferric heme iron. A phosphate group is similar in electronic character to a carboxylate and could give an MCD signal similar to that of a carboxylate-bound heme. Phosphate coordination to the ferric heme iron, albeit trans to imidazole, has recently been seen in a 2.0-Å-resolution crystal structure of imidazole-bound ferric H175G CCP in phosphate buffer [Biochemistry 40 (5) (2001) 1265]. To investigate the validity of phosphate ligation, the pH 5.9 species in the 100 mM phosphate buffer was exchanged into the pH 5.9, 100 mM MES (2-[ N-morpholino]ethanesulfonic acid) buffer. The MCD spectrum of the species in the MES buffer was spectrally distinct from that in the phosphate buffer, indicating that the original spectrum depends on the presence of a phosphate ion. We conclude that in the phosphate buffer, the exogenous ligand-free ferric state of the H175G CCP cavity mutant is actually coordinated by a phosphate ion from the buffer.

Crystal Structure of Rat Heme Oxygenase-1 in Complex with Heme Bound to Azide: IMPLICATION FOR REGIOSPECIFIC HYDROXYLATION OF HEME AT THE α-MESO CARBON

Heme oxygenase (HO) catalyzes physiological heme degradation consisting of three sequential oxidation steps that use dioxygen molecules and reducing equivalents. We determined the crystal structure of rat HO-1 in complex with heme and azide (HO-heme-N(3)(-)) at 1.9-A resolution. The azide, whose terminal nitrogen atom is coordinated to the ferric heme iron, is situated nearly parallel to the heme plane, and its other end is directed toward the alpha-meso position of the heme. Based on resonance Raman spectroscopic analysis of HO-heme bound to dioxygen, this parallel coordination mode suggests that the azide is an analog of dioxygen. The azide is surrounded by residues of the distal F-helix with only the direction to the alpha-meso carbon being open. This indicates that regiospecific oxygenation of the heme is primarily caused by the steric constraint between the dioxygen bound to heme and the F-helix. The azide interacts with Asp-140, Arg-136, and Thr-135 through a hydrogen bond network involving five water molecules on the distal side of the heme. This network, also present in HO-heme, may function in dioxygen activation in the first hydroxylation step. From the orientation of azide in HO-heme-N(3)(-), the dioxygen or hydroperoxide bound to HO-heme, the active oxygen species of the first reaction, is inferred to have a similar orientation suitable for a direct attack on the alpha-meso carbon.

Experimental Observation of Anharmonic Coupling of the Heme-Doming and Iron−Ligand Out-of-Plane Vibrational Modes Confirmed by Density Functional Theory

In the deoxy ferrous state of histidine-ligated heme proteins, the iron−histidine band (νFe-His) has been assigned as a stretching mode that involves a two-body motion involving the iron and histidine combined with a minor amount of heme doming. An analogous Raman band, νFe-L has been observed in the proximal cavity mutant of H93G myoglobin where the Raman band of a series of nonnative axial ligands, L, can be compared. The H93G mutant of myoglobin consists of substitution of the proximal histidine, H93, by glycine. This replacement abolishes the sole covalent connection between the globin and the heme iron and creates a cavity that can be occupied by exogenous ligands, L, by dialysis. In the present study, the iron−axial-ligand out-of-plane vibration, νFe-L, for a series deoxy ferrous heme-iron adducts H93G(L) has been measured as a function of temperature, where L = imidazole (Im), 4-methyl imidazole (4-Me Im), 2-methyl imidazole (2-Me Im), 1-methyl imidazole (1-Me Im), 4-bromo imidazole (4-Br Im), and 2,4-dimethyl imidazaole (2,4-diMe Im). Density functional theory calculations show that anharmonic coupling between low wavenumber heme-doming and iron−ligand out-of-plane modes can account for the calculated νFe-L frequency shifts. These calculations parallel the experimental observed trends for temperature-dependent Raman spectra for all of the adducts except H93G(1-Me Im).

Nature of the displaceable heme-axial residue in the EcDos protein, a heme-based sensor from Escherichia coli.

The EcDos protein belongs to a group of heme-based sensors that detect their ligands with a heme-binding PAS domain. Among these various heme-PAS proteins, EcDos is unique in having its heme iron coordinated at both axial positions to residues of the protein. To achieve its high affinities for ligands, one of the axial heme-iron residues in EcDos must be readily displaceable. Here we present evidence from mutagenesis, ligand-binding measurements, and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopies about the nature of the displaceable residue in the heme-PAS domain of EcDos, i.e., EcDosH. The magnetic circular dichroism spectra in the near-infrared region establish histidine-methionine coordination in met-EcDos. To determine whether in deoxy-EcDos coordination of the sixth axial position is also to methionine, methionine 95 was substituted with isoleucine. This substitution caused the ferrous heme iron to change from an exclusively hexacoordinate low-spin form (EcDosH) to an exclusively pentacoordinate high-spin form (M95I EcDosH). This was accompanied by a modest acceleration of the dissociation rates of ligands but a dramatic increase (60-1300-fold) in the association rate constants for binding of O(2), CO, and NO. As a result, the affinity for O(2) was enhanced 10-fold in M95I EcDosH, but the partition constant M = [K(d)(O(2))/K(d)(CO)] between CO and O(2) was raised to about 30 from the extraordinarily low EcDosH value of 1. Thus a major consequence of the increased O(2) affinity of this sensor was the loss of its unusually strong ligand discrimination.

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O 2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb ( M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20–40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 α-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 ( A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation ( 7.3×10 3 M −1 s −1 ) is similar to that observed for A. thaliana trHbO-3 ( 1.4×10 4 M −1 s −1 ) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN ( 6.7×10 6 M −1 s −1 ). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.

The interplay between heme iron and protein sulfhydryls in the reaction of dimeric Scapharca inaequivalvis hemoglobin with nitric oxide

The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis possesses a single reactive cysteine residue per monomer, Cys92, which is located in the subunit interface in the vicinity of the heme group. The interplay between the heme iron and Cys92 towards the reaction with NO has been investigated by the combined use of electrospray mass spectrometry, FTIR and UV-Visible spectroscopy. When the ferrous liganded or unliganded protein reacts with free NO in solution Cys92 is not modified, but undergoes nitrosation when the hemoglobin is exposed to the nitric oxide releaser S-nitrosocysteine. When the ferric protein reacts with free NO under anaerobic conditions the heme iron is reduced and Cys92 is nitrosated. At variance with other hemeproteins investigated to date, in Scapharca HbI the heme-iron NO driven reduction is not accompanied by the formation of a ferric iron nitrosyl intermediate in detectable amounts. The results are consistent with the hypothesis that the nitrosating agent is the NO + species, which is generated during the NO driven reduction of the ferric heme iron. The possible reaction mechanism is discussed in comparison with recent findings on human hemoglobin and myoglobin.

Raman evidence that the lyoprotectant poly(ethylene glycol) does not restore nativity to the heme active site of horseradish peroxidase suspended in organic solvents.

Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.

Control of Nitric Oxide Synthase Dimer Assembly by a Heme−NO-Dependent Mechanism

Homodimer formation is a key step that follows heme incorporation during assembly of an active inducible nitric oxide synthase (iNOS). In cells, heme incorporation into iNOS becomes limited due to interaction between self-generated NO and cellular heme [Albakri, Q., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 5414-5421]. Here we investigated if NO can regulate at points downstream in the process by inhibiting dimerization of heme-containing iNOS monomer. Heme-containing monomers were generated by treating iNOS dimer or iNOS oxygenase domain dimer (iNOSoxy) with urea. Both monomers dimerized when incubated with Arg and 6R-tetrahydrobiopterin (H4B), as shown previously [Abu-Soud, H. M., Loftus, M., and Stuehr, D. J. (1995) Biochemistry 34, 11167-11175]. The NO-releasing drug S-nitrosyl-N-acetyl-D,L-penicillamine (SNAP; 0-0.5 mM) inhibited dimerization of iNOS monomer in a dose- and time-dependent manner, without causing heme release. SNAP-pretreated monomer also did not dimerize in response to H4B plus Arg. SNAP converted Arg- and H4B-free iNOS dimer into monomer that could not redimerize, but had no effect on iNOS dimer preincubated with Arg and H4B. Anaerobic spectral analysis showed that NO from SNAP bound to the ferric heme of iNOSoxy monomer or dimer. Adding imidazole as an alternative heme ligand prevented SNAP from inhibiting iNOS monomer dimerization. We conclude that NO and related species can block iNOS dimerization at points downstream from heme incorporation. The damage to heme-containing monomer results from a reaction with the protein and appears irreversible. Although dimeric structure alone does not protect, it does enable Arg and H4B to bind and protect. Inhibition appears mediated by NO coordinating to the ferric heme iron of the monomer.

H93G myoglobin cavity mutant as versatile template for modeling heme proteins: magnetic circular dichroism studies of thiolate- and imidazole-ligated complexes.

Recent ligand binding and spectroscopic investigations of the myoglobin H93G cavity mutant are reviewed, revealing it to be a versatile template for the preparation of model heme complexes of defined structure. The H93G myoglobin cavity mutant is shown to be capable of forming mixed ligand adducts because of the difference in accessibility of the two sides of the ferric heme iron. With imidazole bound in the proximal cavity, H93G myoglobin also forms reasonably stable oxyferrous and oxoferryl derivatives, thereby providing a potential system to use for the study of such complexes with proximal ligands other than imidazole. In addition, thiolate-ligated ferric H93G derivatives are described that serve as spectroscopic models for the high-spin ferric state of cytochrome P450. All of the complexes described are characterized with magnetic circular dichroism spectroscopy, and they are compared to the appropriate derivatives of native myoglobin and P450.

Phenylalanine 393 exerts thermodynamic control over the heme of flavocytochrome P450 BM3.

Site-directed mutants of the phylogenetically conserved phenylalanine residue F393 were constructed in flavocytochrome P450 BM3 from Bacillus megaterium. The high degree of conservation of this residue in the P450 superfamily and its proximity to the heme (and its ligand Cys400) infers an essential role in P450 activity. Extensive kinetic and thermodynamic characterization of mutant enzymes F393A, F393H, and F393Y highlighted significant differences from wild-type P450 BM3. All enzymes expressed to high levels and contained their full complement of heme. While the reduction and subsequent treatment of the mutant P450s with carbon monoxide led to the formation of the characteristic P450 spectra in all cases, the absolute position of the Soret absorption varied across the series WT/F393Y (449 nm), F393H (445 nm), and F393A (444 nm). Steady-state turnover rates with both laurate and arachidonate showed the trend WT > F393Y >> F393H > F393A. Conversely, the trend in the pre-steady-state flavin-to-heme electron transfer was the reverse of the steady-state scenario, with rates varying F393A > F393H >> F393Y approximately wild-type. These data are consistent with the more positive substrate-free [-312 mV (F393A), -332 mV (F393H)] and substrate-bound [-151 mV (F393A), -176 mV (F393H)] reduction potentials of F393A and F393H heme domains, favoring the stabilization of the ferrous-form in the mutant P450s relative to wild-type. Elevation of the heme iron reduction potential in the F393A and F393H mutants facilitates faster electron transfer to the heme. This results in a decrease in the driving force for oxygen reduction by the ferrous heme iron, so explaining lower overall turnover of the mutant P450s. We postulate that the nature of the residue at position 393 is important in controlling the delicate equilibrium observed in P450s, whereby a tradeoff is established between the rate of heme reduction and the rate at which the ferrous heme can bind and, subsequently, reduce molecular oxygen.

A photolysis-triggered heme ligand switch in H93G myoglobin.

Resonance Raman spectroscopy and step-scan Fourier transform infrared (FTIR) spectroscopy have been used to identify the ligation state of ferrous heme iron for the H93G proximal cavity mutant of myoglobin in the absence of exogenous ligand on the proximal side. Preparation of the H93G mutant of myoglobin has been previously reported for a variety of axial ligands to the heme iron (e.g., substituted pyridines and imidazoles) [DePillis, G., Decatur, S. M., Barrick, D., and Boxer, S. G. (1994) J. Am. Chem. Soc. 116, 6981-6982]. The present study examines the ligation states of heme in preparations of the H93G myoglobin with no exogenous ligand. In the deoxy form of H93G, resonance Raman spectroscopic evidence shows water to be the axial (fifth) ligand to the deoxy heme iron. Analysis of the infrared C-O and Raman Fe-C stretching frequencies for the CO adduct indicates that it is six-coordinate with a histidine trans ligand. Following photolysis of CO, a time-dependent change in ligation is evident in both step-scan FTIR and saturation resonance Raman spectra, leading to the conclusion that a conformationally driven ligand switch exists in the H93G protein. In the absence of exogenous nitrogenous ligands, the CO trans effect stabilizes endogenous histidine ligation, while conformational strain favors the dissociation of histidine following photolysis of CO. The replacement of histidine by water in the five-coordinate complex is estimated to occur in < 5 micros. The results demonstrate that the H93G myoglobin cavity mutant has potential utility as a model system for studying the conformational energetics of ligand switching in heme proteins such as those observed in nitrite reductase, guanylyl cyclase, and possibly cytochrome c oxidase.

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron.

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.

Effect of rapid heme rotation on electrochemistry of myoglobin

Myoglobins (Mbs) reconstituted with rotatable octamethylheme and non-rotatable etioheme were prepared and their electrochemical behavior was studied. The redox potential of octamethylheme reconstituted Mb (OMe–Mb), of which heme rotates around iron–histidine (F8His) bond, shifted negatively by ca. 30 mV compared with non-rotatable etioheme reconstituted Mb (Etio–Mb). On the other hand, the redox potentials of octamethylheme and etioheme themselves were very similar to each other. Due to the similarity of the distal histidine side of the heme of these two reconstituted Mbs, the shifts of the redox potential would be attributable to the drastic change of the orientation of proximal histidine imidazole ring to the heme plane by heme rotation. The dissociation rate constant of cyanide ion from the ferrous heme iron (II) for OMe–Mb form at 5°C and pH 7.5 was three times faster than that of Etio–Mb. The electron transfer kinetics of these Mbs showed that the heme rotation causes faster electron transfer rates in both electrode reaction and chemical reduction in solution with dithionite. The obtained heterogeneous electron transfer rates constants at an In 2O 3 electrode and first-order rate constants of the chemical reduction were 12(±0.5)×10 −4 cm s −1, 9.8(±1.0) s −1 for OMe–Mb and 6.0(±0.5)×10 −4 cm s −1, 4.5(±1.0) s −1 for Etio–Mb under the present experimental conditions.

Quantum-mechanical tunneling of water in heme proteins

Heme proteins are nanolaboratories that can be used to investigate chemical and biochemical phenomena. For example, the rebinding of carbon monoxide to the Fe2+–protoporphyrin-IX prosthetic group in heme proteins after photodissociation at low temperature (<50 K) proceeds predominantly via quantum-mechanical tunneling, as implied by the presence of an isotope effect on the recombination rate coefficients. Heme proteins are among the few systems where the tunneling of entire molecules has been studied in depth. Recently, we have shown that aquometmyoglobin, which has an Fe3+ heme with a water molecule at the sixth coordination, can be photoreduced at 80 K, yielding a myoglobin with a photodissociable water ligand bound to the ferrous heme iron (Fe2+). We have investigated the rebinding of the water ligand after laser flash excitation using transient absorption spectroscopy with monitoring in the Soret band from 12 to 150 K and 10 ns to 10 s. The kinetics are non-exponential, owing to the heterogeneous nature of the protein ensemble investigated, and are described with a kinetic model involving an enthalpy barrier distribution. Above 50 K, the temperature dependence of the kinetics follows the Arrhenius law, implying that ligand rebinding occurs by classical barrier crossing. Below 50 K, the rebinding occurs markedly faster than predicted from the Arrhenius law, suggestive of molecular tunneling through the barrier. We have used a simple model to describe the kinetics, and fitting of this model to the experimental data allowed us to determine the model parameters governing the tunneling rate coefficient. Copyright © 2000 John Wiley & Sons, Ltd.

Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3.

omega-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium. These compounds are the most potent inhibitors of P-450 BM3 yet reported. All are mixed inhibitors, increasing the Km and decreasing the kcat for laurate oxidation. All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm. Binding to the ferrous form is much weaker. 10-(Imidazolyl)decanoic acid was the best inhibitor (Kic = 0.9 microM, Kiu = 5.7 microM), while 12-(imidazolyl)dodecanoic acid (Kic = 1.35 microM, Kiu = 6.9 microM) was superior to 11-(imidazolyl)undecanoic acid (Kic = 7.5 microM, Kiu = 16 microM). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 microM (C12 azole) and 27 microM (C11 azole). The binding of 10-(imidazolyl)decanoic acid was too tight for an absolute Kd to be determined spectrophotometrically, but this value is <0.2 microM. The binding of different fatty acids to the enzyme was found to have distinct effects on the Kd for the azoles. Laurate induced tighter binding (Kd for the C12 azole lowered to 4.7 microM), while arachidonate weakened the affinity (Kd increased to 23 microM). Arachidonate diminished the affinity for the C10 azole sufficiently that a Kd could be determined by spectrophotometric titration (11 microM). Affinity for the C12 azole was decreased in active-site-mutants R47G (R47 tethers the fatty acid carboxylate group) and F87Y but increased in mutant F87G-indicating an important role for this residue in determining heme accessibility. The C10 azole binds much more weakly to the spin-state-insensitive F87Y (32. 2 microM), suggesting that the inhibitors may bind preferentially to different conformers of P-450 BM3. NADP+ binding in the reductase also tightened affinity of these inhibitors for P-450 BM3 (Kd for the C12 azole decreased to 2.7 microM), but this effect was not observed for FMN-deficient mutant W574D, suggesting that the interdomain effect of NADP+ on inhibitor binding was mediated via flavin mononucleotide. Resonance Raman spectroscopy indicates that the inhibitors form low-spin complexes with P-450 BM3 and that their binding induces movements of the heme vinyls relative to the ring.

Mechanisms of autoxidation of the oxygen sensor FixL and Aplysia myoglobin: implications for oxygen-binding heme proteins.

On exposure to oxygen, ferrous heme is thought to autoxidize via three distinct mechanisms: (i) dissociation of protonated superoxide from oxyheme; (ii) reaction between a noncoordinated oxygen molecule and pentacoordinate deoxyheme, and (iii) reaction between a noncoordinated oxygen molecule and an intermediate having water coordinated to the ferrous heme iron. The formation of a hexacoordinate aquomet (H2O.Fe3+) species has been proposed to drive mechanism (iii); consequently, heme proteins with a pentacoordinate met (Fe3+) form might be expected to lack this pathway. We have measured the dependence of autoxidation rate on oxygen concentration for Rhizobium meliloti FixL and Aplysia kurodai myoglobin, which have pentacoordinate met forms. For both proteins, the bell shape of this dependence shows that they autoxidize primarily by mechanism (iii), indicating that a hexacoordinate aquomet species is not required for this mechanism. A novel presentation of the oxygen dependence of autoxidation rates that uses heme saturation, rather than oxygen concentration, more clearly reveals the relative contributions of autoxidation pathways.

Infrared Crystallography: Structural Refinement through Spectroscopy

A method for determining the orientation of individual bonds within complex macromolecules from polarized IR measurements on oriented single crystals is described. At present, X-ray diffraction is the principal technique used to define the global structure and orientation of macromolecules in the crystalline state. However, resolution limitations and conformational disorder limit the accuracy of the resulting structural model. A quantitative understanding of protein function often requires a more precise description of structural features at a localized active site. Polarized IR measurements of internal stretching bands of N−3, CN−, OCN−, and SCN− bound at the ferric heme iron of single crystals of myoglobin are presented. A synthesis of such measurements on different crystal forms leads to an N−3 orientation consistent with IR measurements on both P21 and P212121 crystals, but significantly different from the orientation reported in X-ray crystal structures. Subtle structural changes between these two crystal forms of the CN− complex may account for small differences in the stretching frequency and orientation of the C–N bond.

Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti

Background: The Rhizobial oxygen sensor FixL is a hemoprotein with kinase activity. On binding of strong-field ligands, a change of the ferrous or ferric heme iron from high to low spin reversibly inactivates the kinase. This spin-state change and other information on the heme pocket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies. We set out to investigate the spin-state of the FixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy. Results: Using one-dimensional N MR we directly observed the high- and low-spin nature of the met- and cyanomet-FixL heme domain, respectively. We determined the hyperfine-shifted 1H-NMR signals of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals. Conclusions: These findings support the spin-state mechanism of FixL regulation. They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent. With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic information about the residues that line the heme pocket.

Inhibition of nitric oxide synthase activity by Zn2+ ion.

We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent Ki of 30 microM. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and also blocks the calmodulin-dependent superoxide-mediated cytochrome c reductase activity exhibited by nNOS. However, Zn2+ ion has no apparent effect on the calmodulin-independent direct reduction of cytochrome c by nNOS. Zn2+ ion induces perturbation difference spectra in nNOS characterized by the appearance of a peak at approximately 430 nm and a trough at approximately 395 nm, with an apparent spectral binding constant of 50 microM. These spectral changes are consistent with a Zn(2+)-dependent change in the spin-state equilibrium of the heme iron in nNOS. The spectral binding constant for L-arginine binding to nNOS (approximately 1.5 microM) is not significantly affected by the presence of 50 microM Zn2+, indicating that Zn(2+)-dependent inhibition of nNOS activity is not due to interference with substrate binding. The estimated maximal change in nNOS absorbance at approximately 418 nm caused by the L-arginine-dependent conversion of the ferric heme iron from hexacoordinate low-spin to pentacoordinate high-spin is increased by 50% in the presence of 50 microM Zn2+, which reflects the increased initial amount of low-spin ferric heme iron present. These data indicate that Zn(2+)-dependent inhibition of nNOS activity is due to binding of Zn2+ to the hemoprotein domain in the enzyme and that inhibition is associated with perturbations in the environment of the heme iron that appear to block its ability to mediate oxygen reduction.