Inhibition of soluble guanylate cyclase by ODQ.

The heme in soluble guanylate cyclases (sGC) as isolated is ferrous, high-spin, and 5-coordinate. [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one] (ODQ) has been used extensively as a specific inhibitor for sGC and as a diagnostic tool for identifying a role for sGC in signal transduction events. Addition of ODQ to ferrous sGC leads to a Soret shift from 431 to 392 nm and a decrease in nitric oxide (NO)-stimulated sGC activity. This Soret shift is consistent with oxidation of the ferrous heme to ferric heme. The results reported here further define the molecular mechanism of inhibition of sGC by ODQ. Addition of ODQ to the isolated sGC heme domain [beta1(1-385)] gave the same spectral changes as when sGC was treated with ODQ. EPR and resonance Raman spectroscopy was used to show that the heme in ODQ-treated beta1(1-385) is indeed ferric. Inhibition of the NO-stimulated sGC activity by ODQ is due to oxidation of the sGC heme and not to perturbation of the catalytic site, since the ODQ-treated sGC has the same basal activity as untreated sGC (68 +/- 12 nmol min(-)(1) mg(-)(1)). In addition, ODQ-oxidized sGC can be re-reduced by dithionite, and this re-reduced sGC has identical NO-stimulated activity as the original ferrous sGC. Oxidation of the sGC heme by ODQ is fast with a second-order rate constant of 8.5 x 10(3) M(-)(1) s(-)(1). ODQ can also oxidize hemoglobin, indicating that the reaction is not specific for the heme in sGC versus that in other hemoproteins.

Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies.

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.

Resonance raman characterization of the heme domain of soluble guanylate cyclase.

We report the resonance Raman characterization of the heme domain of rat lung soluble guanylate cyclase (sGC) expressed in Escherichia coli. Like heterodimeric sGC isolated from bovine lung, the sGC heme domain [beta1(1-385)] and its heme ligand mutant H105G(Im) contain a stoichiometric amount of heme, which is five-coordinate, high-spin ferrous in both beta1(1-385) and chemically reduced H105G(Im). In the presence of NO, both beta1(1-385) and H105G(Im) form a five-coordinate nitrosyl heme complex with a nu(Fe-NO) value of 525 cm-1 and a nu(NO) value of 1676 cm-1. For the first time, the Fe-N-O bending mode near 400 cm-1 has been identified in a five-coordinate nitrosyl heme complex. Both beta1(1-385) and H105G(Im) form a six-coordinate, low-spin complex with CO. We find evidence for two binding conformations of the Fe-CO unit. The conformation that is more prevalent in beta1(1-385) has a nu(Fe-CO) value of 478 cm-1 and a delta(Fe-C-O) value of 567 cm-1, whereas the dominant conformation in H105G(Im) is characterized by a nu(Fe-CO) value of 495 cm-1 and a delta(Fe-C-O) value of 572 cm-1. We propose that in the dominant conformation of H105G(Im)-CO the Fe-CO unit is hydrogen bonded to a distal residue, while this is not the case in beta1(1-385). Reexamination of sGC isolated from bovine lung tissue indicates that it also has two binding conformations for CO; the more populated form is not hydrogen-bonded. We propose that the absence of hydrogen-bond formation between a distal residue and exogenous ligands is physiologically relevant in lowering the oxygen affinity of heterodimeric sGC and, therefore, stabilizing the ferrous, active form of the enzyme under aerobic conditions.

Identification of histidine 105 in the beta1 subunit of soluble guanylate cyclase as the heme proximal ligand.

Soluble guanylate cyclase isolated from bovine and rat lung is a heterodimeric hemoprotein composed of alpha1 and beta1 subunits. The heme binding region has been localized to residues 1-385 of the beta1 subunit [beta1(1-385)], while the catalytic site(s) have been localized to the C-terminal region of sGC. There are four conserved histidine residues in the heme binding region of sGC. H220 and H346 are conserved among all known sGC subunits (alpha and beta), while H105 and H134 are conserved only in the beta subunits (beta1 and beta2). Site-directed mutagenesis was used to individually change each of the conserved histidines in sGC beta1(1-385) to alanine or glycine, and the resulting mutants were expressed in E. coli. All of the mutants except for H105A and H105G had heme bound as isolated. Imidazole (Im) was able to rescue heme binding to H105G when added to the growth medium and purification buffers. The heme in H105G isolated in the presence of imidazole [H105G(Im)] was ferric and a mixture of 5-coordinate, high-spin and 6-coordinate, low-spin complexes. After reduction, the ferrous heme in H105G(Im) was 5-coordinate, high-spin as indicated by resonance Raman spectroscopy. When imidazole in H105G(Im) was exchanged with N-methylimidazole (MeIm), the Fe-N(Im/MeIm) stretching frequency was shifted from 221 to 212 cm-1. A shift of this magnitude is expected when the ligand is directly coordinated to the heme iron. All of the data are consistent with the conclusion that H105 in the beta1 subunit is the heme proximal ligand.

Kok's oxygen clock: What makes it tick? The structure of P680 and consequences of its oxidizing power.

New insights in the structure of P680, the primary electron donor in Photosystem II, are summarized and the implications of its oxidizing power for energy transfer and singlet oxygen production are discussed.