Hemoglobin I (HbI) from the clam Lucina pectinata is an intriguing hemeprotein that binds and transports H 2 S to sulfide-oxidizing chemoautotrophic bacteria to maintain a symbiotic relationship and to protect the mollusk from H 2 S toxicity. Single point mutations at E7, B10 and E11 positions were introduced in the HbI heme pocket to define the reactivity of sulfide with hemeproteins. The functional and structural properties of mutant and wild type recombinant proteins were first evaluated using the well-known ferrous CO and O 2 derivatives. The effects of these mutations on the ferric environment were then studied in the metaquo and hydrogen sulfide derivatives. The results obtained with the ferrous HbI mutants show that all the E7 substitutions and the PheB10Tyr mutation influence directly CO and O 2 binding and stability while the B10 and E11 substitutions induce distal structural rearrangements that affect ligand entry and escape indirectly. For the metaquo-GlnE7His, PheB10Val, PheB10Leu and the E11 variants, two individual distal structures are suggested, one of which is associated with H-bonding interactions between the E7 residues and the bound water. Similar H-bonding interactions are invoked for these HbI-H 2 S mutant derivatives and the rHbI, altering in turn sulfide reactivity within these protein samples. This is evident in the resonance Raman spectra of these HbI-H 2 S complexes, which show reduction of heme iron as judged by the appearance of the ν 4 oxidation state marker at 1356 cm −1 , indicative of heme-Fe II species. This reduction process depends strongly on distal mutations showing faster reduction for those HbI mutants exhibiting strongest H-bonding interactions. Overall, the results presented here show that: a. H 2 S association is regulated by steric constraints; b. H 2 S release is controlled by two competing reactions involving simple sulfide dissociation and heme reduction; c. at high H 2 S concentrations, reduction of the ferric center dominates; d. reduction of the heme is also enhanced in those HbI mutants having polar distal environments. † This work was supported in part by funds from the National Science Foundation, Cellular Biology (Grant 0544250) and NIH-NIGMS/ MBRS-SCORE S06GM08103-34, NCRR G12RR03051, P20RR016439 and NCMHD T37MD001477. ⊥To whom correspondence should be addressed: Telephone (787) Fax (787)265-5476; E-mail E-mail: sonw@caribe.net. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 9. Published in final edited form as:Biochemistry. Hydrogen sulfide (H 2 S) is a well known poisonous gas whose cytotoxic effects have been studied for more than 300 years (1). Interestingly, it has been found recently that H 2 S is produced in mammalian tissues and that, similar to nitric oxide (NO) and carbon monoxide (CO), it may function as a neuromodulator, neuroprotector and/or smooth muscle relaxant/ constrictor (2-5). Endogenous H 2 S is produced at high concentrations in the brain (100 µM) by cystathionine beta-synthase en...

Historically, hydrogen sulfide (H(2)S) has been regarded as a poisonous gas, with a wide spectrum of toxic effects. However, like ·NO and CO, H(2)S is now referred to as a signaling gas involved in numerous physiological processes. The list of reports highlighting the physiological effects of H(2)S is rapidly expanding and several drug candidates are now being developed. As with ·NO and CO, not a single H(2)S target responsible for all the biological effects has been found till now. Nevertheless, it has been suggested that H(2)S can bind to hemeproteins, inducing different responses that can mediate its effects. For instance, the interaction of H(2)S with cytochrome c oxidase has been associated with the activation of the ATP-sensitive potassium channels, regulating muscle relaxation. Inhibition of cytochrome c oxidase by H(2)S has also been related to inducing a hibernation-like state. Although H(2)S might induce these effects by interacting with hemeproteins, the mechanisms underlying these interactions are obscure. Therefore, in this review we discuss the current state of knowledge about the interaction of H(2)S with vertebrate and invertebrate hemeproteins and postulate a generalized mechanism. Our goal is to stimulate further research aimed at evaluating plausible mechanisms that explain H(2)S reactivity with hemeproteins.

The dynamics of the ferric CN complexes of the heme proteins Myoglobin and Hemoglobin I from the clam Lucina pectinata upon Soret band excitation is monitored using infrared and broad band visible pump-probe spectroscopy. The transient response in the UV-vis spectral region does not depend on the heme pocket environment and is very similar to that known for ferrous proteins. The main feature is an instantaneous, broad, short-lived absorption signal that develops into a narrower red-shifted Soret band. Significant transient absorption is also observed in the 360-390 nm range. At all probe wavelengths the signal decays to zero with a longest time constant of 3.6 ps. The infrared data on MbCN reveal a bleaching of the C triple bond N stretch vibration of the heme-bound ligand, and the formation of a five-times weaker transient absorption band, 28 cm(-1) lower in energy, within the time resolution of the experiment. The MbC triple bond N stretch vibration provides a direct measure for the return of population to the ligated electronic (and vibrational) ground state with a 3-4 ps time constant. In addition, the CN-stretch frequency is sensitive to the excitation of low frequency heme modes, and yields independent information about vibrational cooling, which occurs on the same timescale.

Traditionally known as a toxic gas, hydrogen sulfide (H2S) is now recognized as an important biological molecule involved in numerous physiological functions. Like nitric oxide (•NO) and carbon monoxide (CO), H2S is produced endogenously in tissues and cells and can modulate biological processes by acting on target proteins. For example, interaction of H2S with the oxygenated form of human hemoglobin and myoglobin produces a sulfheme protein complex that has been implicated in H2S degradation. The presence of this sulfheme derivative has also been used as a marker for endogenous H2S synthesis and metabolism. Remarkably, human catalases and peroxidases also generate this sulfheme product. In this review, we describe the structural and functional aspects of the sulfheme derivative in these proteins and postulate a generalized mechanism for sulfheme protein formation. We also evaluate the possible physiological function of this complex and highlight the issues that remain to be assessed to determine the role of sulheme proteins in H2S metabolism, detection and physiology.

Several hemoglobins were explored by UV-Vis and resonance Raman spectroscopy to define sulfheme complex formation. Evaluation of these proteins upon the reaction with H2O2 or O2 in the presence of H2S suggest: (a) the formation of the sulfheme derivate requires a HisE7 residue in the heme distal site with an adequate orientation to form an active ternary complex; (b) that the ternary complex intermediate involves the HisE7, the peroxo or ferryl species, and the H2S molecule. This moiety precedes and triggers the sulfheme formation.