Spain will remain under the Trastamaras. One big effect is there won't be any Revolt of Communeros, which was largely caused by Charles V and his appointment of foreigners in Spanish offices. Possible survival of the Princess Isabella and Miguel da Paz (

) who in this case would be future King of Portugal, only.
John as King would certainly have some nice knockoff effects in Italy. He's close to François Ier's age, so you could see the two having some great rivalry akin to François Ier and Charles V. The Habsburg court remains in the Lowlands; possible division of the territories as I did in PoP, but it's not a guarantee. In fact, Charles V will probably end up married to Anna of Bohemia and Hungary and assuming you get a Mohacs like situation, he'd end up King of Bohemia and pursuing claims over Hungary. These alt. Habsburgs would speak French and be based out of Brussels. The Emperor would be in a much stronger position (especially if/when he manages to take over Hungary).
Possibility of butterflies giving Henry VIII a son with Catherine. No great matter. With an Emperor in a stronger position at home (assuming Charles chooses to remain aloof to France's Italian ambitions--I doubt it very much, though) means the Reformation plays out quite differently. Peasants Revolt could be strangled in it's grave, or Charles, in a position of power takes Martin by force from Saxony and has him burned much like John Hus (would cause an uproar, so blatantly invading territory of an Elector, though). You wouldn't strangle Protestantism in the cradle though, but rather radicalize it. You could end up seeing Lutheranism being even more radical than OTL and Calvinism even more so. I dare say if Charles V is as pious as OTL, the HRE will see many years of upheaval and the French may decide to take advantage of that instead of Italy.