Insights into the signal transduction mechanism of RmFixL provided by carbon monoxide recombination kinetics.

This report presents evidence for interdomain steps of the ligand-coupled signal transduction mechanism of the oxygen receptor from Rhizobium meliloti, RmFixL. Photolysis of the CO adducts of heme domain (RmFixLN) and heme kinase (RmFixL*) proteins allowed tracking of second-order heme CO recombination reactions by transient absorbance. Whereas CO rebinding to RmFixLN is characterized by a single kinetic phase, rebinding to RmFixL* is characterized by two kinetic phases. Evidence indicates that CO rebinds to two interconvertible deoxyRmFixL* conformers that are produced sequentially after photolysis. Since the second conformer is only observed when the kinase domain is present, its production is concluded to be an interdomain signal transmission event that is coupled to heme ligand release. Because receptor clustering is a recurring theme in signal transduction mechanisms, the dependence of molecular weight upon heme ligation was investigated at equilibrium. Gel permeation chromatography and native gel electrophoresis showed that the molecular weight distribution for both RmFixLN and RmFixL* depends on heme ligation. At equilibrium, oxyRmFixLN and oxyRmFixL* exist as monomers and dimers, respectively. Their deoxy analogues, metRmFixLN and metRmFixL*, exist as dimers and as a mixture of tetramers and 9-mers, respectively. Assembly of these oligomers is reversible. The physiological relevance of these ligand-coupled assemblies and the kinetic factors controlling CO recombination are discussed.

Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding.

KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.

Nitrosyl adducts of FixL as probes of heme environment.

This report presents a spectroscopic investigation of the nitrosyl adducts of FixL, the sensor in the signal transduction system responsible for regulating nitrogen fixation in Rhizobium meliloti. Variable-temperature resonance Raman (RR), electron spin resonance (ESR), and variable-temperature UV-visible absorption data are presented for the ferrous NO adducts of two FixL deletion derivatives, FixLN (the heme-containing domain) and FixL* (a functional heme-kinase). A temperature-dependent equilibrium is observed between the five-coordinate (5-c) and six-coordinate (6-c) ferrous nitrosyl adducts, with lower temperatures favoring formation of the 6-c nitrosyl adduct. This equilibrium is perturbed as the solution freezes, and the amount of 5-c FixL-NO increases sharply until a nearly constant ratio of 6-c to 5-c adducts is obtained. Complexation between the heme domain of FixL and its response regulator, FixJ, is revealed through specfic FixJ-induced increase in the energy separation between 5-c and 6-c FixL-NO. Ferric nitrosyl adducts of FixL* and FixLN autoreduce to their corresponding ferrous nitrosyl adducts. The kinetic behavior of this reduction is monophasic for FixL*-NO, while the reaction for ferric FixLN-NO is biphasic. These results suggest conformational inhomogeneity in the heme pocket of FixLN and conformational homogeneity in that of FixL*. Hence the kinase domain plays a role in distal pocket conformational stability. Implications for the signal transduction mechanism are discussed.

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant.

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.

Heme speciation in alkaline ferric FixL and possible tyrosine involvement in the signal transduction pathway for regulation of nitrogen fixation.

The pH-dependent behavior of the ferric forms of two soluble truncations of Rhizobium meliloti FixL, FixL (heme and kinase domains, functional), and FixLN (heme domain) are examined by UV-visible, resonance Raman, and electron paramagnetic resonance spectroscopy. Global analysis of UV-visible data indicates that the pKa for hydroxide binding is slightly higher in FixL than in FixLN. Spectroscopic data show that high-spin and low-spin hydroxide adducts of FixLN and FixL exist in a thermal spin-state equilibrium with a significant fraction of the heme in the high spin form at room temperature. FixLN and FixL differ from myoglobin and hemoglobin in that their hemes are not fully ligated by hydroxide ion under strongly alkaline conditions. In addition to the binding of hydroxide ion, both FixLN and FixL undergo additional alkaline transitions that involve the deprotonation of tyrosine residues. FixLN contains four tyrosine residues. One has a pKa of 9.6, which is indistinguishable from that for hydroxide binding to the heme. The other three tyrosines have pKas greater than 11. At pH 11, the alkaline species react with cyanide to yield the familiar low-spin cyanide adduct. Upon reduction of the heme iron, the alkaline forms of the FixL deletion derivatives are converted to their deoxy forms. Resonance Raman spectra reveal that the Fe-His stretching vibrations of deoxyFixLN and deoxyFixL are not measurably shifted from those of their neutral counterparts. Treatment of the alkaline deoxyFixLs with O2 yields the respective oxy forms. Spectroscopic evidence indicates that the loss of activity at elevated pH cannot be attributed solely to generation of a low-spin heme hydroxide. Involvement of one or more tyrosines in signal transmission between the heme and kinase domains of FixL is proposed.

Characterization of ferrous FixL-nitric oxide adducts by resonance Raman spectroscopy.

Resonance Raman spectra of the nitric oxide adducts of the ferrous forms of two soluble truncations of Rhizobium meliloti FixL, FixL* and FixLN, are reported. At room temperature, four isotope sensitive vibrations are observed for both ferrous FixL*-NO and ferrous FixLN-NO. For FixL*-NO, they are observed at 558, 525, 450, and 1675 cm(-1) and are assigned to v(Fe-NO) of a six-coordinate nitrosyl adduct, v(Fe-NO) of a five-coordinate nitrosyl adduct, delta(Fe-NO) of a six-coordinate nitrosyl adduct, and v(N-O) of a five-coordinate nitrosyl adduct, respectively. Similar frequencies are observed for the FixLN-NO isotope sensitive bands. On the basis of the frequencies and spectral separation of the v(Fe-NO) and delta(Fe-NO) modes, the Fe-N-O unit is concluded to have a bent geometry similar to those observed for the nitrosyl adducts of ferrous hemoglobin and myoglobin. Both proteins can be converted to predominantly five-coordinate nitrosyl adducts by lowering the temperature. In low-temperature resonance Raman spectra of FixL*-NO and FixLN-NO, the 558 cm(-1) bands are significantly decreased in intensity and v(Fe-NO)5-c (the Fe-NO stretching vibration for the five-coordinate nitrosyl adduct) is observed at 529 and 526 cm(-1), respectively. Analysis of the v3 and v8 vibrations for these nitrosyl adducts also supports the presence of both five- and six-coordinate nitrosyl adducts of FixL* and FixLN at room temperature and the conversion to predominantly five-coordinate nitrosyl adducts at low temperatures. This temperature-dependent interconversion is reversible. The possible physiological relevance of the thermally accessible five-coordinate state is discussed. The width of v(Fe-NO)6-c at half-height is 1.3 times broader in FixLN-NO than in FixL*-NO, suggesting that the Fe-N-O geometry is more homogeneous in FixL*-NO. In low-temperature spectra of FixLN-NO, a second v(N-O)5-c band is observed, indicating that more than one conformation is attainable in the five-coordinate FixLN-NO. This second v(N-O)5-c is not observed for five-coordinate FixL*-NO, further suggesting a more conformationally restricted nitrosyl heme in FixL*. These variations in the vibrations involving the Fe-NO unit indicate that the kinase domain influences the heme structure. The spectral differences are discussed in terms of the interdomain interactions that result in ligation-dependent mediation of the kinase activity.

Structural basis for ligand discrimination and response initiation in the heme-based oxygen sensor FixL.

FixL is a multiple-domain bacterial O2-sensing protein that modulates the activity of its kinase domain in response to O2 concentration. The kinase activity is coupled, via phosphoryl transfer, to transcriptional activation by a response-regulating protein, FixJ. Heme ligation resulting in a transition from high to low spin inhibits the kinase through an, as yet, ill-defined mechanism. This report presents spectroscopic, kinetic, and thermodynamic data on various complexes of two deletion derivatives of Rhizobium meliloti FixL, FixLN (the heme domain) and a functional heme kinase, FixL*. Resonance Raman characterization of metFixLN and metFixL* indicates that the heme core is smaller than that observed in metmyoglobin and is indicative of a five-coordinate high-spin heme in metFixLs. Resonance Raman spectra of FixL-CO adducts reveal that the Fe-C = O unit and/or its electrostatic environment in FixL*-CO is distorted relative to that in FixLN-CO. The 1H NMR spectra of the met forms further support the model of an asymmetric perturbation of the heme pocket structure associated with the presence of the kinase domain in FixL*. Observation of equivalent Fe-imidazole stretching vibrations for deoxyFixLN and deoxyFixL* (212 cm-1) indicates that the source of this perturbation in the heme pocket of FixL* does not lie on the proximal side of the heme. The equivalent Fe-imidazole stretching frequencies for deoxyFixLN and FixL* indicate that the presence of the kinase domain does not alter the relative strength of the proximal Fe-imidazole bond and that the proximal imidazole ligand is weakly H-bonded, probably to a backbone carbonyl group. Kinetic and thermodynamic data for the reactions of cyanide and fluoride ions with FixL are consistent with shape selectivity due to steric and/or an anisotropic electrostatic field in the distal heme pocket being responsible for the unique reactivities (or lack thereof) of FixL with ligands, i.e., O2, CO, CN-, F-, N3-, and SCN-. While the rate constants for binding of CN- to metFixLN and metFixL* are an order of magnitude slower than that for metMb, the stabilities of these complexes and metMb-CN are nearly the same. Neither N3- nor SCN- binds to the heme with measurable affinity. Since other ferric heme proteins form stable adducts with these ligands, the inability of FixL to form analogous complexes suggests that the ligand selectivity of this protein is rooted in insurmountable activation barriers to the binding of ligands containing more than two atoms and for ligands whose lowest-energy coordination geometries are linear. This allows the natural O2 ligand to compete kinetically with other naturally occurring ligands that form stable complexes with unencumbered hemes. Moreover, the rate constant for binding of CN- to the functional heme-kinase (metFixL*) is smaller than its metFixLN counterpart and the stability of metFixL*-CN is measurably lower than that of metFixLN-CN. This indicates that the contacts between the heme and kinase domains of FixL* impose more stringent geometric constraints on ligand binding than FixLN. The kinase is thus implicated in a possible mechanism for phosphate-dependent feedback control over ligand affinity of the heme.