Characterization of functional heme domains from soluble guanylate cyclase.
Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.
Karow
David S
DS
Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Pan
Duohai
D
Davis
Joseph H
JH
Behrends
Sönke
S
Mathies
Richard A
RA
Marletta
Michael A
MA
D000595:Amino Acid Sequence; D000818:Animals; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016415:Sequence Alignment; D012995:Solubility; D013059:Spectrum Analysis, Raman
D021122:Protein Subunits; D009569:Nitric Oxide; D006418:Heme; D006162:Guanylate Cyclase