Disruption of de novo purine biosynthesis in Pseudomonas fluorescens Pf0-1 leads to reduced biofilm formation and a reduction in cell size of surface-attached but not planktonic cells.

Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25 ∼ 30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.

Systematic analysis of diguanylate cyclases that promote biofilm formation by Pseudomonas fluorescens Pf0-1.

Cyclic di-GMP (c-di-GMP) is a broadly conserved, intracellular second-messenger molecule that regulates biofilm formation by many bacteria. The synthesis of c-di-GMP is catalyzed by diguanylate cyclases (DGCs) containing the GGDEF domain, while its degradation is achieved through the phosphodiesterase activities of EAL and HD-GYP domains. c-di-GMP controls biofilm formation by Pseudomonas fluorescens Pf0-1 by promoting the cell surface localization of a large adhesive protein, LapA. LapA localization is regulated posttranslationally by a c-di-GMP effector system consisting of LapD and LapG, which senses cytoplasmic c-di-GMP and modifies the LapA protein in the outer membrane. Despite the apparent requirement for c-di-GMP for biofilm formation by P. fluorescens Pf0-1, no DGCs from this strain have been characterized to date. In this study, we undertook a systematic mutagenesis of 30 predicted DGCs and found that mutations in just 4 cause reductions in biofilm formation by P. fluorescens Pf0-1 under the conditions tested. These DGCs were characterized genetically and biochemically to corroborate the hypothesis that they function to produce c-di-GMP in vivo. The effects of DGC gene mutations on phenotypes associated with biofilm formation were analyzed. One DGC preferentially affects LapA localization, another DGC mainly controls swimming motility, while a third DGC affects both LapA and motility. Our data support the conclusion that different c-di-GMP-regulated outputs can be specifically controlled by distinct DGCs.

Molecular oxygen regulates the enzymatic activity of a heme-containing diguanylate cyclase (HemDGC) for the synthesis of cyclic di-GMP.

We have studied the structural and enzymatic properties of a diguanylate cyclase from an obligatory anaerobic bacterium Desulfotalea psychrophila, which consists of the N-terminal sensor domain and the C-terminal diguanylate cyclase domain. The sensor domain shows an amino acid sequence homology and spectroscopic properties similar to those of the sensor domains of the globin-coupled sensor proteins containing a protoheme. This heme-containing diguanylate cyclase catalyzes the formation of cyclic di-GMP from GTP only when the heme in the sensor domain binds molecular oxygen. When the heme is in the ferric, deoxy, CO-bound, or NO-bound forms, no enzymatic activity is observed. Resonance Raman spectroscopy reveals that Tyr55 forms a hydrogen bond with the heme-bound O(2), but not with CO. Instead, Gln81 interacts with the heme-bound CO. These differences of a hydrogen bonding network will play a crucial role for the selective O(2) sensing responsible for the regulation of the enzymatic activity.

Protein conformation changes of HemAT-Bs upon ligand binding probed by ultraviolet resonance Raman spectroscopy.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O2) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands, we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free and O2-, CO-, and NO-bound forms of full-length HemAT-Bs and several mutants (Y70F, H86A, T95A, and Y133F) and found that Tyr70 in the heme distal side and Tyr133 and Trp132 from the G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr95 forms a hydrogen bond with heme-bound O2, but Tyr70 does not. It is deduced from this study that hydrogen bonds between Thr95 and heme-bound O2 and between His86 and heme 6-propionate communicate the heme structural changes to the protein moiety upon O2 binding but not upon CO and NO binding. Accordingly, the present UVRR results suggest that O2 binding to heme causes displacement of the G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.

The formation of hydrogen bond in the proximal heme pocket of HemAT-Bs upon ligand binding.

HemAT-Bs is the heme-based O(2) sensor responsible for aerotaxis control in Bacillus subtilis. In this study, we measured the time-resolved resonance Raman spectra of full-length HemAT-Bs wild-type (WT) and Y133F in the deoxy form and the photoproduct after photolysis of CO-bound form. In WT, the nu(Fe-His) band for the 10 ps photoproduct was observed at higher frequency by about 2 cm(-1) compared with that of the deoxy form. This frequency difference is relaxed in hundreds of picoseconds. This time-dependent frequency shift would reflect the conformational change of the protein matrix. On the other hand, Y133F mutant did not show such a substantial nu(Fe-His) frequency shift after photolysis. Since a hydrogen bond to the proximal His induces an up-shift of the nu(Fe-His) frequency, these results indicate that Tyr133 forms a hydrogen bond to the proximal His residue upon the ligand binding. We discuss a functional role of this hydrogen bond formation for the signal transduction in HemAT-Bs.

Crystal structure of CO-sensing transcription activator CooA bound to exogenous ligand imidazole.

CooA is a CO-dependent transcriptional activator and transmits a CO-sensing signal to a DNA promoter that controls the expression of the genes responsible for CO metabolism. CooA contains a b-type heme as the active site for sensing CO. CO binding to the heme induces a conformational change that switches CooA from an inactive to an active DNA-binding form. Here, we report the crystal structure of an imidazole-bound form of CooA from Carboxydothermus hydrogenoformans (Ch-CooA). In the resting form, Ch-CooA has a six-coordinate ferrous heme with two endogenous axial ligands, the alpha-amino group of the N-terminal amino acid and a histidine residue. The N-terminal amino group of CooA that is coordinated to the heme iron is replaced by CO. This substitution presumably triggers a structural change leading to the active form. The crystal structure of Ch-CooA reveals that imidazole binds to the heme, which replaces the N terminus, as does CO. The dissociated N terminus is positioned approximately 16 A from the heme iron in the imidazole-bound form. In addition, the heme plane is rotated by 30 degrees about the normal of the porphyrin ring compared to that found in the inactive form of Rhodospirillum rubrum CooA. Even though the ligand exchange, imidazole-bound Ch-CooA remains in the inactive form for DNA binding. These results indicate that the release of the N terminus resulting from imidazole binding is not sufficient to activate CooA. The structure provides new insights into the structural changes required to achieve activation.

Growth hormone reverses nonalcoholic steatohepatitis in a patient with adult growth hormone deficiency.

BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is an emerging progressive hepatic disease and demonstrates steatosis, inflammation, and fibrosis. Insulin resistance is a common feature in the development of NASH. Molecular pathogenesis of NASH consists of 2 steps: triglyceride accumulation in hepatocytes with insulin resistance and an enhanced oxidative stress caused by reactive oxygen species. Interestingly, NASH demonstrates a striking similarity to the pathologic conditions observed in adult growth hormone deficiency (AGHD). AGHD is characterized by decreased lean body mass, increased visceral adiposity, abnormal lipid profile, and insulin resistance. Moreover, liver dysfunctions with hyperlipidemia and nonalcoholic fatty liver disease (NAFLD) are frequently observed in patients with AGHD, and it is accompanied by metabolic syndrome. METHODS: We studied a case diagnosed as NASH with hyperlipidemia in AGHD. The effect of GH-replacement therapy on the patient was analyzed. RESULTS: Six months of GH-replacement therapy in the patient drastically ameliorated NASH and the abnormal lipid profile concomitant with a marked reduction in oxidative stress. CONCLUSIONS: These results suggest that GH plays an essential role in the metabolic and redox regulation in the liver.

Two ligand-binding sites in the O2-sensing signal transducer HemAT: implications for ligand recognition/discrimination and signaling.

We have identified a ligand (CO) accommodation cavity in the signal transducer sensor protein HemAT (heme-based aerotactic transducer) that allows us to gain single-molecule insights into the mechanism of gas sensor proteins. Specific mutations that are distal and proximal to the heme were designed to perturb the electrostatic field near the ligand that is bound to the heme and near the accommodated ligand in the cavity. We report the detection of a second site in heme proteins in which the exogenous ligand is accommodated in an internal cavity. The conformational gate that directs the ligand-migration pathway from the distal to the proximal site of the heme, where the ligand is trapped, has been identified. The data provide evidence that the heme pocket is the specific ligand trap and suggest that the regulatory mechanism may be tackled starting from more than one position in the protein. Based on the results, we propose a dynamic coupling between the two distinct binding sites as the underlying allosteric mechanism for gas recognition/discrimination that triggers a conformational switch for signaling by the oxygen sensor protein HemAT.

Systematic regulation of the enzymatic activity of phenylacetaldoxime dehydratase by exogenous ligands.

Phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB) contains a heme that acts as the active site for the dehydration reaction of aldoxime. Ferrous heme is the active form, in which the heme is five coordinate with His282 as a proximal ligand. In this work, we evaluated the functional role of the proximal ligand for the catalytic properties of the enzyme by "the cavity mutant technique". The H282G mutant of OxdB lost enzymatic activity, although the heme, which was five coordinate with a water molecule (or OH-) as an axial ligand, existed in the protein matrix. The enzymatic activity was rescued by imidazole or pyridine derivatives that acted as the exogenous proximal ligand. By changing the electron-donation ability of the exogenous ligand with different substituents, the enzymatic activity could be regulated systematically. The stronger the electron-donation ability of the exogenous ligand, the higher was the restored enzymatic activity. Interestingly, H282G OxdB with 2-methyl imidazole showed a higher activity than the wild-type enzyme. Kinetic analyses revealed that the proximal His regulated not only the affinity of substrate binding to the heme but also the elimination of the OH group from the substrate.

Specific hydrogen-bonding networks responsible for selective O2 sensing of the oxygen sensor protein HemAT from Bacillus subtilis.

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector, O2, from other gas molecules to generate the aerotactic signal, but the detailed mechanism of the selective O2 sensing is not obvious. In this study, we measured electronic absorption, electron paramagnetic resonance (EPR), and resonance Raman spectra of HemAT-Bs to elucidate the mechanism of selective O2 sensing by HemAT-Bs. Resonance Raman spectroscopy revealed the presence of a hydrogen bond between His86 and the heme propionate only in the O2-bound form, in addition to that between Thr95 and the heme-bound O2. The disruption of this hydrogen bond by the mutation of His86 caused the disappearance of a conformer with a direct hydrogen bond between Thr95 and the heme-bound O2 that is present in WT HemAT-Bs. On the basis of these results, we propose a model for selective O2 sensing by HemAT-Bs as follows. The formation of the hydrogen bond between His86 and the heme propionate induces a conformational change of the CE-loop and the E-helix by which Thr95 is located at the proper position to form the hydrogen bond with the heme-bound O2. This stepwise conformational change would be essential to selective O2 sensing and signal transduction by HemAT-Bs.

Recognition and discrimination of gases by the oxygen-sensing signal transducer protein HemAT as revealed by FTIR spectroscopy.

The determination of ligand binding properties is a key step in our understanding of gas sensing and discrimination by gas sensory proteins. HemAT is a newly discovered signal transducer heme protein that recognizes O(2) and discriminates against other gases such as CO and NO. We have used FTIR spectroscopy on CO- and NO-bound sensor domain HemAT and sensor domain distal mutants Y70F, T95A, R91A, and L92A to gain insight into the structure of the iron-bound ligand at ambient temperature. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand and also allow us to investigate the communication pathway between the distal residues of the protein and the heme. We show the formation of both H-bonded and non-H-bonded conformations in the CO-bound forms. In addition, we report the presence of multiple conformations in the NO-bound forms. Such distal H-bonding is crucial for ligand binding and activation by the heme. The comparison of the O(2), NO, and CO data demonstrates that Thr95 and Tyr70 are crucial for ligand recognition and discrimination and, thus, for specific sensing of gases, and L92 is crucial for controlling the conformational changes of the Thr95 and Tyr70 residues upon NO binding.

Crystallization and preliminary X-ray analysis of CooA from Carboxydothermus hydrogenoformans.

CooA, a homodimeric haem-containing protein, is responsible for transcriptional regulation in response to carbon monoxide (CO). It has a b-type haem as a CO sensor. Upon binding CO to the haem, CooA binds promoter DNA and activates expression of genes for CO metabolism. CooA from Carboxydothermus hydrogenoformans has been overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion method. The crystal belongs to space group P2(1), with unit-cell parameters a = 61.8, b = 94.7, c = 92.8 angstroms, beta = 104.8 degrees. The native and anomalous difference Patterson maps indicated that two CooA dimers are contained in the asymmetric unit and are related by a translational symmetry almost parallel to the c axis.

Raman evidence for specific substrate-induced structural changes in the heme pocket of human cytochrome P450 aromatase during the three consecutive oxygen activation steps.

Specific substrate-induced structural changes in the heme pocket are proposed for human cytochrome P450 aromatase (P450arom) which undergoes three consecutive oxygen activation steps. We have experimentally investigated this heme environment by resonance Raman spectra of both substrate-free and substrate-bound forms of the purified enzyme. The Fe-CO stretching mode (nu(Fe)(-)(CO)) of the CO complex and Fe(3+)-S stretching mode (nu(Fe)(-)(S)) of the oxidized form were monitored as a structural marker of the distal and proximal sides of the heme, respectively. The nu(Fe)(-)(CO) mode was upshifted from 477 to 485 and to 490 cm(-)(1) by the binding of androstenedione and 19-aldehyde-androstenedione, substrates for the first and third steps, respectively, whereas nu(Fe)(-)(CO) was not observed for P450arom with 19-hydroxyandrostenedione, a substrate for the second step, indicating that the heme distal site is very flexible and changes its structure depending on the substrate. The 19-aldehyde-androstenedione binding could reduce the electron donation from the axial thiolate, which was evident from the low-frequency shift of nu(Fe)(-)(S) by 5 cm(-)(1) compared to that of androstenedione-bound P450arom. Changes in the environment in the heme distal site and the reduced electron donation from the axial thiolate upon 19-aldehyde-androstenedione binding might stabilize the ferric peroxo species, an active intermediate for the third step, with the suppression of the formation of compound I (Fe(4+)=O porphyrin(+)(*)) that is the active species for the first and second steps. We, therefore, propose that the substrates can regulate the formation of alternative reaction intermediates by modulating the structure on both the heme distal and proximal sites in P450arom.

Spectroscopic and substrate binding properties of heme-containing aldoxime dehydratases, OxdB and OxdRE.

Aldoxime dehydratase (Oxd) is a novel hemeprotein that catalyzes the dehydration reaction of aldoxime to produce nitrile. In this study, we studied the spectroscopic and substrate binding properties of two Oxds, OxdB from Bacillus sp. strain OxB-1 and OxdRE from Rhodococcus sp. N-771, that show different quaternary structures and relatively low amino acid sequence identity. Electronic absorption and resonance Raman spectroscopy revealed that ferric OxdRE contained a six-coordinate low-spin heme, while ferric OxdB contained a six-coordinate high-spin heme. Both ferrous OxdRE and OxdB included a five-coordinate high-spin heme to which the substrate was bound via its nitrogen atom for the reaction to occur. Although the ferric Oxds were inactive for catalysis, the substrate was bound to the ferric heme via its oxygen atom in both OxdB and OxdRE. Electronic paramagnetic resonance (EPR) and rapid scanning spectroscopy revealed that the flexibility of the heme pocket was different between OxdB and OxdRE, which might affect their substrate specificity.

Evidence for displacements of the C-helix by CO ligation and DNA binding to CooA revealed by UV resonance Raman spectroscopy.

The UV and visible resonance Raman spectra are reported for CooA from Rhodospirillum rubrum, which is a transcriptional regulator activated by growth in a CO atmosphere. CO binding to heme in its sensor domain causes rearrangement of its DNA-binding domain, allowing binding of DNA with a specific sequence. The sensor and DNA-binding domains are linked by a hinge region that follows a long C-helix. UV resonance Raman bands arising from Trp-110 in the C-helix revealed local movement around Trp-110 upon CO binding. The indole side chain of Trp-110, which is exposed to solvent in the CO-free ferrous state, becomes buried in the CO-bound state with a slight change in its orientation but maintains a hydrogen bond with a water molecule at the indole nitrogen. This is the first experimental data supporting a previously proposed model involving displacement of the C-helix and heme sliding. The UV resonance Raman spectra for the CooA-DNA complex indicated that binding of DNA to CooA induces a further displacement of the C-helix in the same direction during transition to the complete active conformation. The Fe-CO and C-O stretching bands showed frequency shifts upon DNA binding, but the Fe-His stretching band did not. Moreover, CO-geminate recombination was more efficient in the DNA-bound state. These results suggest that the C-helix displacement in the DNA-bound form causes the CO binding pocket to narrow and become more negative.

Gene expression profile in the heart of spontaneous dwarf rat: in vivo effects of growth hormone.

Excess and deficit of growth hormone (GH) both affect cardiac architecture as well as its function. To date, experimental and clinical studies have reported that GH has an inotropic effect on animal and human heart, however, it remains controversial whether GH is applicable to the treatment for the patients with chronic heart failure. Also, the mechanism by which GH exerts these biological effects on the heart is not well understood. In this study, we attempted to specify the genes regulated by GH in the heart of spontaneous dwarf rat using a microarray analysis. We found that soluble forms of guanylate cyclase, cofilin1, and thymosin beta4 mRNA were up-regulated in the heart by GH treatment. On the other hand, acyl-CoA synthetase, aldosterone receptor, myosin regulatory light chain, troponin T, laminA, and beta-actin mRNA were down-regulated. These results suggest GH regulates essential molecules that regulate structural, contractile, remodeling, and regenerative functions. Collectively, our data indicate a new integrative understanding for the biological effects of GH on cardiac function.

Biophysical properties of a c-type heme in chemotaxis signal transducer protein DcrA.

Chemotaxis signal transducer protein DcrA from a sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was previously shown to contain a c-type heme in its periplasmic domain (DcrA-N) for sensing redox and/or oxygen [Fu et al. (1994) J. Bacteriol. 176, 344-350], which is the first example of a heme-based sensor protein containing a c-type heme as a prosthetic group. Optical absorption and resonance Raman spectroscopies indicates that heme c in DcrA-N shows a redox-dependent ligand exchange. Upon reduction, a water molecule that may be the sixth ligand of the ferric heme c is replaced by an endogenous amino acid. Although the reduced heme in DcrA-N is six-coordinated with two endogenous axial ligands, CO can easily bind to the reduced heme to form CO-bound DcrA-N. Reaction of the reduced DcrA-N with molecular oxygen results in autoxidation to form a ferric state without forming any stable oxygen-bound form probably due to the extremely low redox potential of DcrA-N (-250 mV). Our study supports the initial idea by Fu et al. that DcrA would act as a redox and/or oxygen sensor, in which the ligand exchange between water and an endogenous amino acid is a trigger for signal transduction. While the affinity of CO to DcrA-N (Kd = 138 microM) is significantly weak compared to those of other heme proteins, we suggest that CO might be another physiological effector molecule.

Absence of a detectable intermediate in the compound I formation of horseradish peroxidase at ambient temperature.

A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H(2)O(2) by HRP is significantly faster than 1 x 10(4) s(-1).

Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex.

Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nM, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (approximately 3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.

Structural diversities of active site in clinical azole-bound forms between sterol 14alpha-demethylases (CYP51s) from human and Mycobacterium tuberculosis.

To gain insights into the molecular basis of the design for the selective azole anti-fungals, we compared the binding properties of azole-based inhibitors for cytochrome P450 sterol 14alpha-demethylase (CYP51) from human (HuCYP51) and Mycobacterium tuberculosis (MtCYP51). Spectroscopic titration of azoles to the CYP51s revealed that HuCYP51 has higher affinity for ketoconazole (KET), an azole derivative that has long lipophilic groups, than MtCYP51, but the affinity for fluconazole (FLU), which is a member of the anti-fungal armamentarium, was lower in HuCYP51. The affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 was quite low compared with that to HuCYP51. In the resonance Raman spectra for HuCYP51, the FLU binding induced only minor spectral changes, whereas the prominent high frequency shift of the bending mode of the heme vinyl group was detected in the KET- or 4-PhIm-bound forms. On the other hand, the bending mode of the heme propionate group for the FLU-bound form of MtCYP51 was shifted to high frequency as found for the KET-bound form, but that for 4-PhIm was shifted to low frequency. The EPR spectra for 4-PhIm-bound MtCYP51 and FLU-bound HuCYP51 gave multiple g values, showing heterogeneous binding of the azoles, whereas the single gx and gz values were observed for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectroscopic differences suggest that the region between the B' and C helices, particularly the hydrophobicity of the C helix, in CYP51s plays primary roles in determining strength of interactions with azoles; this differentiates the binding specificity of azoles to CYP51s.