Chemical structure of HistidineImage:Histidine_resonant.png
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Histidine is one of the 20 most common natural amino acids present in proteins. In the nutritional sense, in humans, histidine is considered an essential amino acid, but mostly only in children. The imidazole side chains and the relatively neutral pK of histidine (ca 6.0) mean that relatively small shifts in cellular pH will change its charge. For this reason, this amino acid side chain finds its way into considerable use as a co-ordinating ligand in metalloproteins, and also as a catalytic site in certain enzymes. more...

Heparin sodium
Hexal Diclac
Hexal Ranitic

The imidazole side chain has two nitrogens with different properties: One is bound to hydrogen and donates its lone pair to the aromatic ring and as such is slighty acidic, whereas the other one donates only one electron to the ring so it has a free lone pair and is basic. These properties are exploited in different ways in proteins. In catalytic triads, the basic nitrogen of histidine is used to abstract a proton from serine, threonine or cysteine to activate it as a nucleophile. In a histidine proton shuttle, histidine is used to quickly shuttle protons, it can do this by abstracting a proton with its basic nitrogen to make a positively-charged intermediate and then use another molecule, a buffer, to extract the proton from its acidic nitrogen. In carbonic anhydrases, a histidine proton shuttle is utilized to rapidly shuttle protons away from a zinc-bound water molecule to quickly regenerate the active form of the enzyme.

The amino acid is a precursor for histamine and carnosine biosynthesis.

There are two isoforms: D-histidine and L-histidine.


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Role of the coordinating histidine in altering the mixed valency of Cu(A): An electron nuclear double resonance-electron paramagnetic resonance investigation
From Biophysical Journal, 5/1/02 by Lukoyanov, Dmitriy

ABSTRACT The binuclear CU^sub A^ site engineered into Pseudomonas aeruginosa azurin has provided a CU^sub A^-azurin with a

well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to CU^sub A^, notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C^sub beta^ protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence CUA systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the CUA core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His,120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear CU^sub A^ center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.


Cu^sub A^ in cytochrome c oxidase was recognized even in the 1970s as having unusual electronic properties (Beinert, 1997), for example, by its small histidine and Cys C^sub beta^ proton hyperfine couplings (Stevens et al., 1982; van Camp et al., 1978) and by its small copper hyperfine couplings resolved at S-band (Froncisz et al., 1979). Subsequently, the marked similarities between the Cu^sub A^ center of cytochrome oxidase and the binuclear copper of nitrous oxide reductase in their low frequency, seven-line EPR pattern (Antholine, 1997; Karpefors et al., 1996; Kroneck et al., 1990) and in the considerable homology of sequence near the copper binding sites (Zumft et al., 1992; Zumft and Kroneck, 1996) all pointed to CU^sub A^ as a binuclear, mixed-- valence [Cu(1.5)...Cu(1.5)] center. Mixed-valency will arise when there is a large exchange coupling that delocalizes an unpaired valence electron throughout the CuSSCu core and when this coupling is larger than vibronic, Jahn-Teller-like interactions or any local copper site differences that tend to valence-trap spin on one of the coppers (Farrar et al., 1996; Schatz, 1980). The purple Cu^sub A^ center was firmly established as a binuclear, mixed-valence complex through biophysical (Blackburn et al., 1994; Farrar et al., 1995; Greenwood et al., 1983; Kroneck et al., 1990), biochemical (Dennison et al., 1995; Hay et al., 1996; Stutter et al., 1996; van der Oost et al., 1992), and x-ray crystallographic study (Iwata et al., 1995; Robinson et al., 1999; Tsukihara et al., 1995; Williams et al., 1999; Wilmanns et al., 1995). The Cu^sub A^ center (called Cu^sub A^-azurin hereafter) as engineered by loop-directed mutagenesis into Pseudomonas aeruginosa azurin (Hay et al., 1996, 1998) provided high resolution (1.65 A) to the structure of Cu^sub A^ in a protein, whereby bond distances to weaker axial ligands and to equatorial histidine ligands were obtained along with the tilt of the histidine imidazole planes. (A schematic of the locale of the Cu^sub A^ centers from wild-type CU^sub A^-azurin is shown in Fig. 1.) One histidine ligand (Hisl20/H120) conspicuously showed both a longer bond distance and a larger out-of-plane tilt than the other histidine (His46/H46), and the copper that coordinated His120 also happened to be the recipient of a short bond to a carbonyl oxygen, whose Cu-oxygen bond length was little different from the His120-to-Cu bond distance. The existence of two detectable and structurally slightly different CuA centers within the wild-type Cu^sub A^-azurin crystal with different Cu-His120 bond lengths was resolved, as indicated in Fig. 1. Wild-type CUA-azurin showed the typical 7-line EPR pattern characteristic of mixed-valence [Cu(1.5)..Cu(1.5)] copper (Hay et al., 1998).

As observed from crystal structures of recombinant and native CU^sub A^ systems (Iwata et al., 1995; Tsukihara et al., 1995; Wilmanns et al., 1995; Robinson et al., 1999), the axial ligand distance, Cu-Cu bond distance, and equatorial histidine nitrogen bonding are interdependent (Farrar et al., 1996, Gamelin et al., 1998). Furthermore, they are coupled to the electron transfer function of the site by their affect on the reduction potential, bonding, reorganization energy, and electronic structure of the copper center. In an effort to modulate the bonding of the equatorial ligand, and thus vary the axial ligand interaction and Cu-Cu bonding, His 120 was mutated to asparagine and glycine in Cu^sub A^ azurin (Wang et al., 1999; Berry et al., 2000).

ENDOR-EPR measurements are relevant to this biological electron transfer function and bonding because they provide an experimental measure of electron delocalization and an experimental measure of impaired spin density on the cysteine and histidine ligands through which electron transfer may occur. Multifrequency EPR and Q-band ENDOR studies were done to identify the underlying electronic structural character of Cu^sub A^-azurin and its His120 mutants and to identify the role of His 120 in altering this electronic structural character (Berry et al., 2000; Wang et al., 1999).


The CU^sub A^-azurin, its Hisl20Asn (H120N) and Hisl20Gly (H120G) mutants, and Type 1 azurin per se were expressed, prepared, and characterized as described previously (Hay et al., 1996; Wang et al., 1999). The concentration of these samples was ~1 mM, and they were dissolved in 50% glycerol, 0.1 M phosphate buffer, pH 5.2. Beef heart cytochrome C (aa3) oxidase was prepared as described previously (Fan et al., 1988). Copper-- substituted liver alcohol dehydrogenase in the presence of NADH (Maret and Kozlowski, 1987) was prepared by C. T. Martin. ENDOR experiments were carried out at Q-band (Sienkiewicz et al., 1996) under cryogenic, pumped helium rapid passage conditions as outlined previously (Veselov et al., 1998). Cryogenic multifrequency EPR spectra at S, C, and X-band were taken and analyzed at the National Biomedical ESR Center (Antholine, 1997). Loop-gap resonators and low frequency microwave bridges designed and built at the National Biomedical ESR Center (Medical College of Wisconsin, WI) were used at S- and C-band (Froncisz and Hyde, 1982). The temperature for multifrequency EPR was controlled with a helium flow system (Air Products, Allentown, PA). A Gaussmeter (Rawson-Lush Instrument, Inc., Acton, MA) was used to calibrate the magnetic field and an EIP Model 548 frequency counter used to measure the microwave frequency.


The wild-type CU^sub A^-azurin system, although engineered into a Type 1 copper azurin through loop-directed mutagenesis, showed intimate electronic structure and hyperfine information from its cysteines, histidine, and copper, which are highly typical of the CU^sub A^ of naturally occurring systems. The characteristics of the Cys C^sub beta^ proton and histidine hyperfine couplings that reflected specific covalency of the CuSSCu core, as well as the weak proton couplings that qualitatively reflected overall spin distribution, remained essentially unchanged among CuC^sub A^-azurin and its H120N and H120G mutants. The ENDOR findings and UV-Vis (Berry et al., 2000; Wang et al., 1999) information would imply that the core electronic structure remains unchanged among Cu^sub A^-azurin and its H120N and H120G mutants. Multifrequency EPR at similar cryogenic temperatures to those of ENDOR showed that the His120 mutations led to an increase in gi (2.17-2.23) and a copper hyperfine pattern that diminished from the seven lines expected from mixed-- valence copper to no more than four copper features in a manner that might be expected from trapped-valence mononuclear copper. These results indicate that the His120 ligand plays a subtle role in modulation of electronic structure of the Cu^sub A^ center and different spectroscopic techniques are required to fully understand the effect of the mutation.

If the magnitude of the copper hyperfine coupling and the presence of fewer lines indicates smaller spin density and only one copper, then there would have to be a vast rearrangement of electron spin density for the His120 mutants. One could postulate that while one copper was losing spin, the other could concurrently gain it in the course of valence localization; the net population of spin on the cysteine sulfur could then conceivably stay constant in the process. However, this explanation would also need to postulate a change of conformation of the remaining histidine (His46) to explain why the nitrogen hyperfine coupling to that nonmutated histidine, which should be located next to the copper with spin to account for hyperfine coupling to 14 N of His46, should stay constant. Conversely, if it were to be supposed that the His120 mutants are mixed-valent, then there would need to be a marked change in the Cu hyperfine coupling for one copper, while the spin density throughout the overall CuSSCu site remained unchanged. We are left with a puzzle for the His120 mutants to explain both the constancy of the overall electronic spin distribution implied by ENDOR and the localization of spin on one copper implied by multifrequency EPR. The explanation of the puzzle may require theory at the present state of the art (Gamelin et al., 1998; Neese, 1997), and indeed be a test of that theory, to accurately calculate ligand and metal Fermi and dipolar hyperfine contributions for a CuA that explicitly lacks one histidine.

This work was supported by the National Institutes of Health Grant GM-35103 (to C.P.S.) and National Science Foundation CHE95-02421 (to Y.L.). W.E.A. was supported by National Institutes of Health RRO1008 to J.S. Hyde. Y.L. also acknowledges the Camille and Henry Dreyfus Foundation for the Camille Dreyfus Teacher-Scholar award. We are grateful to Dr. C.T. Martin, now Prof. of Chemistry, University of Massachusetts, Amherst, for providing the CuLADH sample made in the presence of NADH.


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Dmitriy Lukoyanov,* Steven M. Berry,^ Yi Lu,^ William E. Antholine,^^ and Charles P. Scholes*

*Department of Chemistry and Center for Biological Macromolecules, University at Albany, SUNY, Albany, New York 12222 USA;

^Department of Chemistry, Chemical & Life Sciences Laboratory, University of Illinois, Urbana, Illinois 61801 USA; and

^^National Biomedical Electron Spin Resonance Center, Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 USA

Submitted November 17, 2001, and accepted for publication February 13, 2002.

Dr. Lukoyanov is on leave from the MRS Laboratory, Kazan State University, 420008, Kazan, Russian Federation.

Address reprint requests to Dr. Charles P. Scholes, Department of Chemistry, University at Albany, SUNY, Albany, NY 12222. Tel: 518-442-- 4551; Fax: 518-442-3462; E-mail: cps14@

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